Coumarin compounds suitable for inhibiting occurrence of cell death and application of coumarin compounds

Abstract

The invention relates to an application of a group of furocoumarin compounds in preparation of a medicine for inhibiting diseases caused by cell death. The furocoumarin compounds are white angelica, white angelica brain, alloimperatorin, imperatorin, hydrated oxidized imperatorin or decuricin. The invention also provides application of the furocoumarin compound in preparation of an experimental reagent for inhibiting diseases caused by cell death. Experiments show that imperatorin and hydrated oxidized imperatorin can significantly improve the survival rate of influenza lethal model mice. The hydrated oxypeucedanin can significantly reduce the fatality rate of cytokine storm syndrome model mice. The invention illustrates that the furocoumarin compound inhibits cell death, delays or blocks the occurrence of cytokine storm, further reduces the development of acute lung injury or multi-organ function failure, and has considerable clinical application prospects.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and more specifically, to a group of coumarin compounds suitable for inhibiting cell death and their applications. Background Technology

[0002] Cell death and proliferation are crucial for maintaining homeostasis. Among programmed cell death pathways, pyroptosis, apoptosis, and necroptosis are the most typical. Different cell death pathways can induce complex inflammatory responses through different cascade signaling. Cytokine storm (CS) is a life-threatening systemic inflammatory state characterized by elevated levels of circulating cytokines and excessive activation of immune cells. CS is considered one of the leading causes of death in patients infected with influenza virus, SARS-CoV, and MERS-CoV. Studies have shown a direct correlation between CS and acute lung injury, multiple organ failure, and poor prognosis. One possible mechanism between CS and organ damage is inflammatory cell death. A 2021 article by Karki et al. published in *Cell* found that during SARS-CoV-2 infection, only the combination of TNF-α and IFN-γ induced inflammatory cell death characterized by pyroptosis, apoptosis, and necroptosis. When researchers administered neutralizing antibodies against TNF-α and IFN-γ, the survival rate of mice in various lethal mouse models was significantly improved, including a TNF-α and IFN-γ-induced inflammatory shock model, a SARS-CoV-2 infection lethal model, a poly I:C+LPS lethal model, and an LPS lethal model. Therefore, targeting and inhibiting TNF-α and IFN-γ-induced inflammatory cell death is an effective means of blocking CS (cytokine scarring).

[0003] Furanocoumarins are widely found in nature. Traditional Chinese medicines such as Angelica dahurica, Angelica pubescens, Peucedanum praeruptorum, Angelica sinensis, and Psoralea corylifolia, as well as many other medicinal plants reported abroad, all contain furanocoumarin compounds. Furanocoumarins exhibit a wide range of pharmacological activities, primarily including antitumor, anti-HIV, antioxidant, photochemical, antimicrobial, anti-inflammatory, analgesic, antidepressant, and effects on drug-metabolizing enzymes. Byakangelicin, Byakangelicol, Alloimperatorin, Imperatorin, Oxypeucedaninhydrate, and Nodakenin are all natural furanocoumarins extracted from medicinal plants and possess broad-spectrum pharmacological potential. Studies have reported that Byakangelicin can improve carbon tetrachloride-induced liver fibrosis and liver damage by inhibiting hepatic stellate cell proliferation and activation, and inhibiting hepatocyte apoptosis. Angelica sinensis has been shown to inhibit tumor cell proliferation and has antioxidant effects. Current research suggests that imperatorin possesses clear anti-tumor properties, exhibiting anti-cancer activity in cervical cancer cells HeLa, SiHa, and MS-75, and inducing apoptosis in HeLa cells. Similarly, imperatorin also shows anti-tumor and tumor metastasis-inhibiting properties, inhibiting the proliferation of fibroblast-like synovial cells in rheumatoid arthritis and inducing their apoptosis. Studies have found that imperatorin can reduce the production of IL-6 and TNF-α induced by yeast polysaccharides. Preliminary studies on hydrated oxidized imperatorin show anti-tumor cell proliferation and antiviral activity. Imperatorin can induce breast cancer cell death. In summary, current research on six furanocoumarin compounds mainly focuses on their anti-tumor cell proliferation, antioxidant, or in vitro antiviral effects.

[0004] However, the intervention effects of yakangelicin, byakangelicol, alloimperatorin, imperatorin, oxypeucedanin hydrate, and nodakenin on cell death have not been reported. Therefore, elucidating their inhibitory effects on cell death, delaying or blocking the occurrence of cytokine storms, and thus reducing the development of acute lung injury or multiple organ failure, holds considerable clinical application potential. Summary of the Invention

[0005] The purpose of this invention is to address the shortcomings of the prior art by providing a new use for a group of coumarin compounds suitable for inhibiting cell death.

[0006] In a first aspect, the present invention provides the application of a group of furanocoumarin compounds in the preparation of drugs for inhibiting cell death in diseases, wherein the furanocoumarin compounds are angelica lin, angelica natifida, imperatorin, imperatorin, hydrated oxyimperatorin, or imperatorin.

[0007] As a preferred example, the cell death occurs in diseases such as severe viral pneumonia, systemic inflammatory response syndrome, sepsis, graft-versus-host disease, or hemophagocytic lymphohistiocytosis.

[0008] As a preferred example, the compound is prepared into a pharmaceutical formulation by adding pharmaceutically acceptable excipients and a carrier, wherein the excipients include at least one of fillers, disintegrants, binders, excipients, diluents, lubricants, sweeteners, and colorants.

[0009] Secondly, the present invention provides the application of a group of furanocoumarin compounds in the preparation of experimental reagents for inhibiting cell death in diseases, wherein the furanocoumarin compounds are angelica linolenic acid, angelica linolenic acid, imperatorin, imperatorin, hydrated oxyimperatorin, and imperatorin.

[0010] As a preferred example, the diseases in which cell death occurs are severe viral pneumonia, systemic inflammatory response syndrome, sepsis, graft-versus-host disease, or hemophagocytic lymphohistiocytosis.

[0011] The advantages of this invention are as follows: Experiments have shown that both imperatorin and hydrated oxyimperatorin can significantly improve the survival rate of influenza-induced fatal mouse models. Hydrated oxyimperatorin can significantly reduce the proportion of peripheral blood leukocytes in mice, decrease the number of peripheral blood erythrocytes, hemoglobin content, hematocrit, platelets, and platelet-weighted hematocrit, reduce the level of serum alanine aminotransferase in mice, and inhibit the occurrence of cytokine storms caused by TNF-α+IFN-γ attack-induced cell death. Byakangelicin, Byakangelicol, Alloimperatorin, Imperatorin, Oxypeucedanin hydrate, and Nodakenin have intervention effects on cell death. This invention elucidates that furanocoumarin compounds inhibit cell death, delay or block the occurrence of cytokine storms, and thus reduce the development of acute lung injury or multiple organ failure, showing considerable clinical application potential. Attached Figure Description

[0012] Figure 1 Structure of coumarin compounds.

[0013] Figure 2The toxicity of different coumarin compounds to macrophages.

[0014] Figure 3 The inhibitory effects of different coumarin compounds on macrophage death.

[0015] Figure 4 Microscopic observation of the inhibitory effects of different coumarin compounds on macrophage death.

[0016] Figure 5 Survival rate of mice in a PR8 virus infection-induced mortality model. A. Results of in vivo intervention with different doses of imperatorin; B. Results of in vivo intervention with different doses of hydrated oxypereratorin.

[0017] Figure 6 The protective effect of hydrated oxidized imperatorin on a mouse model of cytokine storm syndrome. A. Hydrated oxidized imperatorin reduced the mortality rate of the model mice; the results were obtained from two independent replicate experiments. B. Flow cytometry was used to detect the proportion of leukocytes in the peripheral blood of mice. C. A hematology analyzer was used to detect the number of erythrocytes, hemoglobin content, hematocrit, platelets, and platelet-weighted hematocrit in the peripheral blood of mice. D. Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were detected. Detailed Implementation

[0018] The present invention will be further described below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the description of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0019] Example 1

[0020] 2.1 Structure of Coumarin Compounds

[0021] Byakangelicin, Byakangelicol, Alloimperatorin, Imperatorin, Oxypeucedaninhydrate, Nodakenin Figure 1 .

[0022] 2.2 Cell viability assay

[0023] Method: Take 6 ～Eight-week-old SPF-grade male C57BL / 6 mice were sacrificed and disinfected by immersion in 75% alcohol. The femur and tibia were isolated and placed in BMDM (bone marrow-derived mononuclear macrophage) complete culture medium (1640 medium containing 10% FBS, 1% penicillin and streptomycin, and 1% non-essential amino acids). A 100μm sterile filter was placed on a 50mL centrifuge tube, pre-wetted with complete culture medium, and then the femur and tibia were cut off at both ends with ophthalmic scissors. The contents of the bone marrow cavity were flushed onto the filter with complete culture medium until the bone turned white. The contents of the bone marrow cavity were ground using the tail of a 1mL syringe, and complete culture medium was added. The mixture was centrifuged at 1500rpm for 5 minutes, the supernatant was discarded, and 1 mL of the culture medium was added. ～ Resuspend in 2 mL of erythrocyte lysis buffer, 2 ～ Centrifuge at 1500 rpm for 5 minutes after 3 minutes, then wash the cells once more with PBS. Plate the cells into 10 cm diameter cell culture dishes (2 mice / dish) and incubate for 2 hours. ～ After 3 hours, gently aspirate the cell supernatant using a disposable pipette (you can lightly rinse the culture dish to improve cell yield). Centrifuge at 1500 rpm for 5 minutes and discard the supernatant. Resuspend the cells in complete culture medium. Add 10 μL of the cell suspension to 10 μL of trypan blue staining reagent, dilute 2-fold, and count cells using a hemocytometer. Adjust the cell concentration to 2 × 10⁶ cells / mL. 7 Cells were seeded per well in 24-well plates, and M-CSF was added to the complete culture medium to a final concentration of 20 ng / mL. The plates were incubated at 37°C in a 5% CO2 incubator on day 0. The cell culture medium was changed on days 2 and 4. On day 6, the cells were treated with 0-1600 μM of the drug, and cell viability was assessed using the CCK8 assay after 24 h of treatment.

[0024] Prepare CCK8 working solution by adding 10 μL of CKK8 to every 100 μL of DMEM complete culture medium. Remove all culture medium from the test wells, add 110 μL of CCK8 working solution to each well, and incubate in a cell culture incubator at 37℃ and 5% CO2 for 30-60 min. Observe the color change of the solution; it can be detected when it turns light orange-yellow. Keep away from light. Use a multi-mode microplate reader to detect the OD value of each well at 450 nm, and calculate the cell viability of each well using the formula.

[0025] Results: All compounds showed no toxicity at concentrations below 100 μM, and subsequent intervention doses were set below 100 μM. Figure 2 ).

[0026] 2.3 Coumarin compounds inhibit macrophage death

[0027] Methods: BMDM cells were pretreated with six coumarin compounds for 2 h. A control group (1 mL of 1640 medium per well) and a dexamethasone group (Dex, 10 μg / mL) were also established. After 2 hours of incubation, TNF-α (50 ng / mL) + IFN-γ (100 ng / mL) were added for stimulation. After 48 hours of further incubation, cells were collected for analysis. Macrophage death was detected by flow cytometry using propidium iodide (PI) staining.

[0028] Results: Except for Oxypeucedanin hydrate at 12.5 μM, Byakangelicin, Byakangelicin, Alloimperatorin, Imperatorin, and Nodakenin at concentrations of 6.25 μM and 12.5 μM significantly improved the survival of BMDM cells and inhibited BMDM cell death. Figure 3 The protective effect of the compound on the survival of BMDM cells was also observed under a microscope. Figure 4 ).

[0029] 2.4 Imperatorin and hydrated oxyimperatorin reduced the mortality rate of influenza virus-infected mice in vivo.

[0030] Methods: ① Experimental grouping: Fifty-eight SPF-grade C57BL6 / J mice were divided into four groups: model group (n=12), imperatorin 15 mg / kg / d (n=12), imperatorin 50 mg / kg / d (n=12), hydrated oxidized imperatorin 15 mg / kg / d (n=11), and hydrated oxidized imperatorin 50 mg / kg / d (n=11). ② Mouse treatment and infection: Mice were randomly assigned to cages and acclimatized for 5 days before being inhaled with influenza A virus A / PR8 / 34 (PR8). On the day of infection, mice were anesthetized by intraperitoneal injection and then inhaled with a diluted PR8 virus solution, 40 μL / mouse, at a dose of 70 PFU / mouse. Two hours after infection, the corresponding dose of the drug was injected intraperitoneally. The model group was injected with a drug-free working solution twice a day, 8 hours apart, 200 μL each time, for 5 days. The weight and mortality of mice were recorded daily after infection, and the observation continued for 20 days.

[0031] Results: The survival rate of mice in the model group was 25%. The survival rate of mice treated with imperatorin 15 mg / kg / d was 41.7%, the survival rate of mice treated with imperatorin 50 mg / kg / d was 66.7%, the survival rate of mice treated with hydrated oxidized imperatorin 15 mg / kg / d was 63.6%, and the survival rate of mice treated with hydrated oxidized imperatorin 50 mg / kg / d was 81.8%. Both imperatorin and hydrated oxidized imperatorin significantly improved the survival rate of influenza-induced death model mice.

[0032] 2.5 Hydrated oxidized imperatorin reduced the lethality of a mouse model of cytokine storm syndrome in vivo.

[0033] 2.5.1 Hydrated oxidized imperatorin can significantly reduce the mortality rate of mice in a cytokine storm syndrome model.

[0034] Methods: Fourteen SPF-grade C57BL6 / J mice were randomly divided into a model group (n=7) and a hydrated oxidized imperatorin group (4 mg / kg) (n=7). After 5 days of acclimatization, the mice were challenged with intraperitoneal injection of TNF-α + IFN-γ (15 + 30 μg). Two hours later, the hydrated oxidized imperatorin group received a single intraperitoneal injection of 4 mg / kg. The survival of the mice was then observed every half hour for a total observation period of 30 hours.

[0035] Results: The survival rate of mice in the model group was 14.3%, while the survival rate of mice in the hydrated oxidized imperatorin group after a single dose was 57.1%; hydrated oxidized imperatorin significantly reduced the lethality of mice in the cytokine storm syndrome model. Figure 6 A).

[0036] 2.5.2 Hydrated oxidized imperatorin inhibits the occurrence of cytokine storm in mice caused by TNF-α+IFN-γ attack-induced cell death.

[0037] Methods: Fifteen SPF-grade C57BL6 / J mice were randomly divided into a blank control group (n=5), a model group (n=5), and a hydrated oxidized imperatorin group (8 mg / kg / d) (n=5). After 5 days of acclimatization, the mice were challenged with intraperitoneal injection of TNF-α + IFN-γ (15 + 30 μg). 0.5 h before challenge, the hydrated oxidized imperatorin group received a single intraperitoneal injection of 8 mg / kg, while the model group received a drug-free working solution. 7 h after challenge, peripheral blood was collected from the mice. Flow cytometry was used to detect the proportion of CD45+ leukocytes in peripheral blood; a hematology analyzer was used to detect the number of erythrocytes, hemoglobin content, hematocrit, platelets, and platelet-weighted hematocrit; and the levels of alanine aminotransferase (ALT, C009-2-1) and aspartate aminotransferase (AST, C010-2-1) in the mouse serum were detected using the Nanjing Jiancheng Biotechnology Institute test kit.

[0038] Results: Hydrated oxidized imperatorin significantly reduced the proportion of peripheral blood leukocytes in mice, decreased the number of peripheral blood erythrocytes, hemoglobin content, hematocrit, platelets, and platelet-weighted hematocrit, reduced the level of serum alanine aminotransferase in mice, and inhibited the occurrence of cytokine storm in mice caused by TNF-α+IFN-γ attack-induced cell death. Figure 6 BD).

[0039] The above description is only a preferred embodiment of the present invention. It should be noted that those skilled in the art can make several improvements and additions without departing from the method of the present invention, and these improvements and additions should also be considered within the scope of protection of the present invention.

Claims

1. The application of a group of furanocoumarin compounds in the preparation of drugs for diseases that inhibit cell death, characterized in that, The furanocoumarin compounds are angelicin, angelicin, imperatorin, imperatorin, hydrated oxyimperatorin, or imperatorin.

2. The application according to claim 1, characterized in that, The diseases in which cell death occurs include severe viral pneumonia, systemic inflammatory response syndrome, sepsis, graft-versus-host disease, or hemophagocytic lymphohistiocytosis.

3. The application according to claim 1 or 2, characterized in that, The compound is prepared into a pharmaceutical formulation by adding pharmaceutically acceptable excipients and a carrier, wherein the excipients include at least one of fillers, disintegrants, binders, excipients, diluents, lubricants, sweeteners, and colorants.

4. The application of a group of furanocoumarin compounds in the preparation of experimental reagents for inhibiting cell death in diseases, characterized in that, The furanocoumarin compounds are angelicin, angelicin, imperatorin, imperatorin, hydrated oxyimperatorin, and imperatorin.

5. The application according to claim 4, characterized in that, The diseases in which cell death occurs include severe viral pneumonia, systemic inflammatory response syndrome, sepsis, graft-versus-host disease, or hemophagocytic lymphohistiocytosis.

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