Application of I-type interferon receptor antagonist and / or signaling pathway inhibitor in preparation of medicine for preventing and treating heatstroke

Abstract

The invention discloses application of an I-type interferon receptor antagonist and / or a signaling pathway inhibitor in preparation of a medicine for preventing and treating heatstroke. Whole genome CRISPR / Cas9 screening finds that an I-type interferon signal channel is significantly activated in cells subjected to heat stress, and qPCR experiments prove that molecules of the I-type interferon signal channel are highly expressed, indicating that the I-type interferon signal channel participates in multi-organ injury after heatstroke. The use of the IFNAR1 neutralizing antibody and / or the I-type interferon signal channel inhibitor can reduce heat stress cell death, and reduce death rate and multi-organ injury of heatstroke mice, which indicates that the IFNAR1 antagonist and / or the I-type interferon signal channel inhibitor can play a role in protecting multiple organs after heatstroke by inhibiting heat stress cell injury and death, and can be used for preventing heatstroke. The clinical development and application prospects are realized.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology and relates to the use of a type I interferon receptor antagonist and / or a type I interferon receptor antagonist and / or a signal pathway inhibitor in the preparation of a drug for preventing and treating heatstroke; in particular, it relates to the use of the drug in the preparation of a drug for preventing, treating or alleviating severe heatstroke / multiple organ damage after heatstroke. Background Art

[0002] Heatstroke refers to an acute disease characterized by central nervous system and (or) cardiovascular dysfunction as the main manifestations due to dysfunction of the body temperature regulation center, failure of sweat gland function and (or) excessive loss of water and electrolytes in an environment with high temperature and (or) high humidity. Depending on the severity of the symptoms, heatstroke can be divided into pre-heatstroke, mild heatstroke and severe heatstroke. The incidence of severe heatstroke has been on the rise in recent years, with a mortality rate as high as 60%. In terms of the prevention and treatment of heatstroke, the main measures currently include timely cooling, replenishing water and electrolytes, and protecting organ functions, but there is a lack of specific drugs and unified treatment plans. The current bottleneck in the prevention and treatment of heatstroke is that the understanding of the fatal mechanism of severe heatstroke is still not completely clear, resulting in difficulty in blocking the progression of the disease even with timely cooling.

[0003] When a person experiences heatstroke, their core body temperature may exceed 40°C, causing damage to macromolecular structures such as intracellular proteins, DNA, and lipid membranes, disrupting the cytoskeleton and organelles, and ultimately triggering cell death. This cell damage and death releases a large amount of inflammatory mediators, further exacerbating the systemic inflammatory response.

[0004] The classic type I interferon signaling pathway is initiated as follows: Type I interferons (such as interferon α and interferon β) bind to type I interferon receptors (IFNARs) on the cell surface. IFNARs are composed of two subunits, IFNAR1 and IFNAR2. Type I interferons bind to the receptors and activate the IFNAR1-associated tyrosine kinase TYK2 and the IFNAR2-associated tyrosine kinase JAK1. Activated JAK1 and TYK2 phosphorylate tyrosine residues on the receptor subunits, which in turn phosphorylate STAT1 and STAT2. Phosphorylated STAT1 and STAT2 bind to IRF9 to form the ISGF3 complex. The ISGF3 complex translocates to the nucleus, binds to the interferon-stimulated response element (ISRE), and regulates the expression of interferon-stimulated genes (ISGs), such as ZBP1.

[0005] Type I interferons (such as interferon β) can induce the expression of the necroptosis-related gene Z-DNA binding protein 1 (ZBP1), thereby promoting the occurrence of cell necroptosis.

[0006] Currently, there is no effective drug for preventing and treating multiple organ damage after heatstroke. Therefore, it is urgent to clarify the factors that cause and develop multiple organ damage after heatstroke and explore drug treatments to prevent and treat multiple organ damage after heatstroke. Summary of the Invention

[0007] The present invention aims to provide the use of type I interferon receptor (IFNAR1) antagonists and / or type I interferon body antagonists and / or signaling pathway inhibitors in the preparation of drugs for preventing and treating heatstroke. By inhibiting heat stress-induced activation of the type I interferon signaling pathway, the present invention, using experimental models of different cell types and mice, has demonstrated that type I interferon receptor (IFNAR1) antagonists and / or type I interferon signaling pathway inhibitors can inhibit cell damage and death after heat stress, thereby alleviating multi-organ damage after heatstroke and reducing heatstroke mortality.

[0008] The object of the present invention is achieved through the following technical solutions:

[0009] The present invention provides use of a type I interferon receptor antagonist and / or a type I interferon signaling pathway inhibitor in preparing a medicine for preventing, treating or alleviating heatstroke.

[0010] In the present invention, the heatstroke includes pre-heatstroke, mild heatstroke (such as heat cramps, heat syncope, etc.) and severe heatstroke (such as heat stroke, heat exhaustion, etc.), as well as diseases and symptoms caused by increased body temperature.

[0011] As an embodiment of the present invention, the type I interferon receptor antagonist is at least one selected from antibodies, polypeptides and small molecules.

[0012] As one embodiment of the present invention, the type I interferon receptor antagonist is an antibody; and the antibody is selected from at least one of a monoclonal anti-type I interferon receptor antibody and a monoclonal anti-type 1 interferon antibody.

[0013] As one embodiment of the present invention, the type I interferon receptor antagonist includes a type I interferon receptor antibody: InVivoMab anti-mouse IFNAR1 (BE0241). It is a monoclonal antibody for blocking mouse type I interferon receptor (IFNAR1), clone number: MAR1-5A3, and can be used to block the IFNAR1 signaling pathway in vivo and in vitro.

[0014] As one embodiment of the present invention, the type I interferon antagonist includes an antagonist that targets and blocks the human type I interferon signaling pathway.

[0015] As an embodiment of the present invention, the antagonist targeting and blocking the human type I interferon signaling pathway is selected from at least one of the following: Anifrolumab, Sifalimumab, and anti-IFN-α monoclonal antibody Rontalizumab.

[0016] Anifrolumab (which specifically binds to IFNAR1 and blocks type I interferon); currently used to treat patients with moderate to severe systemic lupus erythematosus and systemic sclerosis.

[0017] Sifalimumab (Anti-IFNa1) is a fully human monoclonal antibody used to target and inhibit human interferon α (IFN-α). It inhibits abnormal immune activity by binding to multiple IFN-α subtypes.

[0018] Rontalizumab (anti-IFN-α monoclonal antibody); is a humanized IgG1 monoclonal antibody designed to bind to and neutralize human IFN-α subtypes, blocking their interaction with the type I interferon receptor (IFNAR).

[0019] As an embodiment of the present invention, the type I interferon signaling pathway inhibitor includes at least one of the STAT1 inhibitor fludarabine, the JAK1 inhibitor filgotinib, and abrocitinib.

[0020] The present invention has the following beneficial effects:

[0021] The present invention relates to cell death induced by activation of the interferon signaling pathway in cells caused by heat stress. The use of IFNAR1 antagonists or interferon signaling pathway inhibitors can protect cells after heat stress and reduce multi-organ damage after heat stroke. The invention can be used to prepare drugs for preventing and / or treating severe heat stroke / heatstroke. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Other features, objects and advantages of the present invention will become more apparent upon reading the detailed description of non-limiting embodiments with reference to the following drawings:

[0023] Figure 1 This is a volcano plot (Figure A) of differentially expressed genes that regulate heat stress cell death using whole-genome screening using CRISPR / Cas9 technology, and a map of the enrichment of the type I interferon signaling pathway (Figure B).

[0024] Figure 2 Schematic diagram showing that heat stress increases the IFN-β content in MLE12 cells (Figure A) and the expression of type I interferon-stimulated genes (ISGs) (Figure B).

[0025] Figure 3Schematic diagram of IFNAR1 neutralizing antibodies improving the cell survival rate of BEND3 cells (Figure A) and L929 cells (Figure B) after heat stress (the results shown in the figure are mean ± standard deviation, n = 4).

[0026] Figure 4 Schematic diagram of the effect of STAT1, an inhibitor of the type I interferon signaling pathway molecule, on improving the cell survival rate of L929 cells after heat stress (the results shown in the figure are mean ± standard deviation, n = 5).

[0027] Figure 5 Schematic diagram of the increased serum IFN-β content in heatstroke model mice.

[0028] Figure 6 Schematic diagram showing that IFNAR1 gene knockout improves the cell survival rate of BMDM cells after heat stress; A is an immunoblot image; B is a schematic diagram of the survival rate of BMDM cells after heat stress.

[0029] Figure 7 Schematic diagram of the survival rates of normal mice and IFNAR1 gene knockout mice in a heat stroke model.

[0030] Figure 8 Serum biochemical results of multiple organ damage in normal mice and IFNAR1 gene knockout mice after heat stroke model (results shown in the figure are mean ± standard deviation, n = 4).

[0031] Figure 9 These are the lung tissue pathology images of WT mice and IFNAR1 gene knockout mice under normal conditions and after heatstroke model. DETAILED DESCRIPTION

[0032] The present invention will be described in detail below with reference to the examples. The following examples will help those skilled in the art to further understand the present invention, but are not intended to limit the present invention in any form. It should be noted that those skilled in the art may make several adjustments and improvements without departing from the scope of the present invention. These all fall within the scope of protection of the present invention.

[0033] Experimental animals: Male 8-week-old wild-type C57 / BL6 mice and IFNAR1 knockout mice (C57 / BL6 strain background) were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.

[0034] Experimental cells: MLE12 cells and L929 cells were purchased from ATCC, and BEND3 cells were purchased from Prosai. Primary bone marrow macrophages (BMDM) were extracted and induced from wild-type C57 / BL6 mice and IFNAR1 gene knockout mice.

[0035] BMDM cell extraction and differentiation induction: The mice were killed by cervical dislocation and disinfected by soaking in 75% alcohol for 5 minutes. The hind limbs were separated, the muscle tissue was removed, and the femur and tibia were completely removed. Bone marrow flushing: Cut the two ends of the bone, use a 1mL syringe to absorb pre-cooled PBS, flush the bone marrow into a 15mL centrifuge tube, and flush repeatedly until the bone head turns white. Cell dispersion and filtration: Blow the bone marrow suspension until there are no clumps, pass it through a 40μm cell sieve, centrifuge (300×g, 5 minutes) and discard the supernatant. Red blood cell lysis: Add 3mL ACK buffer, lyse on ice for 2 minutes, centrifuge and resuspend in DMEM complete medium. Cell culture and differentiation, initial inoculation: use 1×10 6 Seed cells in culture plates at a density of 10 cells / mL. Culture in complete DMEM medium supplemented with M-CSF (10 ng / ml) at 37°C, 5% CO2. Medium exchange and observation: Day 3: Change medium halfway (retain adherent cells). Days 5-7: Observe that mature macrophages are round or polygonal with prominent pseudopodia. Remove M-CSF and proceed with experimental treatments.

[0036] Experimental Materials: The CCK-8 kit was purchased from Tongren Biotechnology (Cat. No. CK04); the RNA reverse transcription kit (Cat. No. RR036A) and the fluorescent quantitative PCR kit (Cat. No. RR420L) were purchased from Takara; the IFNAR1 neutralizing antibody anti-IFNAR1 (InVivoMab anti-mouse IFNAR-1 (BE0241)) and the isotype control antibody anti-IgG (InVivoMab IgG1 isotype control (BE0088)) were purchased from Bio X Cell. The STAT1 inhibitor Fludarabine (F-ara-A; NSC 118218) was purchased from MCE (Cat. No. HY-B0069). Primers for IFN-β, ISG15, Cxcl9, IRF7, MX1, Oasl2, and ZBP1 mRNA were synthesized by Shanghai Sangon Biotechnology Technology Service Co., Ltd. (primer sequences are shown in the table below).

[0037] Table 1

[0038]

[0039]

[0040] Cell culture: MLE12, L929, and BEND3 cells were cultured in DMEM (complete DMEM) supplemented with 10% fetal bovine serum, 100 U / ml penicillin, and 100 mg / L streptomycin. Primary bone marrow macrophages (BMDMs) were cultured in complete DMEM supplemented with M-CSF (10 ng / mL) to induce differentiation. Cells were cultured in a cell culture incubator at 37°C and 5% CO2. During heat stress, cells were cultured in a cell culture incubator at 42-43°C and 5% CO2.

[0041] Preparation of cell heat stress injury model: MLE12 cells, L929 cells, and BEND3 cells were cultured normally for 12 hours, and then the cell culture plates were placed in a cell culture incubator at 43°C and 5% CO2 for heat stress culture for 3 hours; primary bone marrow macrophages (BMDM) were induced with M-CSF for 5 days and then placed in a cell culture incubator at 43°C and 5% CO2 for heat stress culture for 2 hours; then the cell culture plates were immediately returned to a cell culture incubator at 37°C and 5% CO2 for recovery. Cells were collected at different time points to extract RNA and detect the mRNA levels of related molecules, or the CCK-8 kit was used to detect cell activity after 12 hours of recovery.

[0042] Preparation of mouse heatstroke model: The mice were placed in a constant humidity and temperature control box at a temperature of 40℃±0.3℃ and a humidity of 60±5% for heat stress for 180 minutes (laboratory preliminary experiments found that the core body temperature of 8-week-old C57 / BL6 mice rose to 42℃ after about 180 minutes), and then immediately removed from the temperature control box and placed in an environment of 22℃ and 35% humidity for normal breeding.

[0043] Biomolecular Experimental Methods: For experimental methods in the examples where specific conditions are not specified, conventional conditions were generally followed, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or the conditions recommended by the kit manufacturer.

[0044] Statistical Analysis: All numerical variables in this study are expressed as mean ± standard error. Two-group comparisons were performed with the two-tailed Student's t-test, three-group comparisons were performed with the one-way ANOVA test, and four-group comparisons were performed with the two-way ANOVA and Sidák's multiple comparisons test. Statistically significant differences were considered when P < 0.05. * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, and *** indicates P < 0.0001. If no indicator line is drawn, the comparison is compared with the control group (con group).

[0045] Example 1. Construction and screening of whole-genome CRISPR / Cas9 lentiviral library cells

[0046] Construction of MLE12 cells stably expressing Cas9: The Cas9-expressing LentiCRISPRv2-mCherry plasmid (Addgene Plasmid #99154) and packaging plasmids psPAX2 and pMD2.G were transfected into HEK293T cells at a mass ratio of 2:2:1 to package lentivirus expressing Cas9 and mCherry. MLE12 cells were infected with this lentivirus. After 24 hours, single cells were sorted using a SONY flow cytometer to obtain MLE12 cells stably expressing Cas9.

[0047] Construction of MLE12-CRISPR / Cas9 whole genome library cells: The mouse whole genome gRNA library plasmid GeWKOv2 (Addgene Plasmid #67988) and packaging plasmids psPAX2 and pMD2.G were co-transfected into HEK293T cells at a mass ratio of 2:2:1 to package the library virus. In 10,000 MLE12 cells stably expressing Cas9, different volume gradients of virus (0 μL, 2 μL, 4 μL, 6 μL and 8 μL) were set to explore the optimal volume of virus-infected cells. 8 μg / mL polybrene was supplemented and infected for 24 hours. The infection efficiency was detected by BV421 fluorescence flow cytometry, and the virus volume with a cell infection rate of 30% was selected for subsequent infection. Culture 1×10 8 MLE12-Cas9 cells were infected with the library virus, and the infected positive cells were sorted using a SONY flow cytometer after 24 hours.

[0048] Screening experiment: After the obtained library cells were stably passaged, they were subjected to heat stress modeling at 43°C for 3 hours, and then placed in a normal temperature incubator at 37°C for 24 hours. The medium was changed and the living cells were collected after washing with PBS. The library cells that did not experience heat stress were used as the control group. DNA was extracted for high-throughput sequencing. The molecules involved in regulating heat stress cell death were analyzed based on the retained sgRNA, and GSEA analysis was performed. The results are attached. Figure 1 As shown in the figure, IFNAR1 molecule ranked high in the screening results, and GSEA analysis results showed that type I interferon signaling pathway molecules were enriched in heat stressed cells.

[0049] Example 2: IFN-β and ISGs are highly expressed in heat-stressed cells

[0050] Preparation of cell heat stress injury model: After culturing MLE12 cells normally for 12 hours, the cell culture plate was placed in a cell culture incubator at 43°C and 5% CO2 for 2 hours of heat stress culture; then the cell culture plate was immediately returned to a cell culture incubator at 37°C and 5% CO2 to recover. At different time points, cells were collected to extract RNA and detect the mRNA levels of IFN-β and ISGs-related molecules. The results are shown in the attached figure. Figure 2 As shown, the mRNA expression of IFN-β gene continued to increase from 6 hours to 24 hours after cell heat stress, and the mRNA expression of interferon-stimulated genes (ISGs) such as ZBP1 increased.

[0051] Example 3: IFNAR1 neutralizing antibodies improve the survival of heat-stressed cells

[0052] After L929 cells and BEND3 cells were cultured normally for 12 hours, IFNAR1 neutralizing antibody (anti-IFNAR1) or isotype control IgG antibody (anti-IgG) was added. After treating the cells for 8 hours, the cell culture plates were placed in a cell culture incubator at 43°C and 5% CO2 for heat stress culture for 3 hours; then the cell culture plates were immediately returned to a cell culture incubator at 37°C and 5% CO2. After recovery for 12 hours, the cell activity was detected using a CCK-8 kit to evaluate the degree of cell heat stress damage. The results are shown in the attached Figure 3 As shown, anti-IFNAR1 can promote the survival of L929 cells and BEND3 cells after heat stress, that is, reduce the damage and death of heat stressed cells.

[0053] Example 4: Type I interferon signaling pathway inhibitors can inhibit heat stress cell death

[0054] One hour before heat stress, 10 nM of STAT1 inhibitor Fludarabine (referred to as STAT1 inhibitor in the attached figure) was added to the L929 cells. An equal volume of solvent DMSO was added to the control group. Then, a heat stress model was established as in Example 3 and cell activity was detected using a CCK-8 kit to evaluate the degree of cell damage after heat stress. The results are shown in the attached figure. Figure 4 As shown in the results, STAT1 inhibitors can significantly improve the cell survival rate of L929 cells after heat stress and inhibit heat stress damage.

[0055] Example 5: Increased IFN-β levels in the serum of mice with heat stroke

[0056] Eight-week-old male wild-type C57 / BL6 mice were randomly divided into two groups. One group was housed in a normal environment at 22°C (Control group); the other group was subjected to a heatstroke model (Heat group). After 6 hours of recovery, orbital venous blood was collected from the mice and the IFN-β content was detected using an ELISA kit. The results are shown in the attached figure. Figure 5 As shown, the IFN-β content in the serum of heatstroke mice was increased.

[0057] Example 6: IFNAR1 gene knockout can inhibit heat stress cell death

[0058] BMDM were extracted from the bone marrow of wild-type C57 / BL6 mice and IFNAR1 knockout mice, respectively, and labeled as WT group and IFNAR1 KO group. Cell activity was detected using CCK-8 kit before heat stress. Then, the cell heat stress injury model was prepared according to the above method. After 12 hours of recovery, cell activity was detected again using CCK-8. The results are shown in the attached Figure 6 Figure A shows that immunoblotting experiments proved that IFNAR1 gene knockout was successful, and Figure B showed that IFNAR1 gene knockout can increase the survival rate of BMDM cells after heat stress and reduce the degree of damage.

[0059] Example 7: IFNAR1 gene knockout improves the survival rate of mice in a heat stroke model

[0060] Wild-type C57 / BL6 mice and IFNAR1 knockout mice were randomly divided into 4 groups. + / + group), wild-type C57 / BL6 mouse heatstroke model group (Heat-IFNAR1 + / + group), IFNAR1 gene knockout mouse control group (Shame-IFNAR1 - / - group), IFNAR1 gene knockout mouse heatstroke model group (Heat-IFNAR1 - / -group), 5 rats in each control group, and 15 rats in each heatstroke model group (the mortality rate of heatstroke model is high).

[0061] Blank control group (Shame-IFNAR1 + / + 、Shame-IFNAR1 - / - ): Normally raised in a 22℃ environment.

[0062] Heat stroke model group (Heat-IFNAR1 + / + 、Heat-IFNAR1 - / - ): Heat stress was applied for 180 minutes in a constant humidity and temperature control box at a temperature of 40°C ± 0.3°C and a humidity of 60 ± 5% (laboratory preliminary experiments showed that the core body temperature of 8-week-old male C57 / BL6 mice increased to 42°C after about 180 minutes). The mice were then immediately removed from the temperature control box and placed in a 22°C environment for normal feeding.

[0063] Survival rate analysis: Count the number of mice that died and the time point, and make a survival curve for heat stroke model mice. The results are attached. Figure 7 The survival rate of IFNAR1 gene knockout mice was significantly higher than that of wild-type mice.

[0064] Analysis of multiple organ damage: Heat stroke model mice were allowed to recover in a normal feeding environment for 8 hours, then anesthetized with sodium pentobarbital injection, orbital venous blood was collected, and serum biochemical indicators were tested. The results are shown in the attached Figure 8 IFNAR1 gene knockout significantly inhibited liver, kidney and myocardial damage in heatstroke model mice.

[0065] Observation of lung tissue pathological damage: After collecting orbital venous blood as described above, mice were perfused with systemic circulation, and then lung tissue was removed and sliced ​​for HE staining. Figure 9 , Heat-IFNAR1 + / + In the lung tissue of mice in the group, mild thickening of the alveolar wall, congestion of alveolar capillaries, infiltration of inflammatory cells, and expression of Heat-IFNAR1 were observed. - / - (IFNAR1 gene knockout) mice showed reduced damage.

[0066] In summary, the present invention provides the use of type I interferon receptor antagonists and / or signaling pathway inhibitors in the preparation of drugs for preventing or treating heatstroke. The present invention found through whole-genome CRISPR / Cas9 screening that the type I interferon signaling pathway was significantly activated in cells experiencing heat stress, and the high expression of molecules in the type I interferon signaling pathway was confirmed by qPCR experiments, indicating that the type I interferon signaling pathway is involved in multiple organ damage after heatstroke. The present invention uses IFNAR1 neutralizing antibodies and type I interferon signaling pathway inhibitors to reduce heat stress cell death, reduce the mortality rate and multiple organ damage in mice with heatstroke, indicating that IFNAR1 antagonists and / or type I interferon signaling pathway inhibitors can play a protective role on multiple organs after heatstroke by inhibiting heat stress cell damage and death, and have clinical development and application prospects.

[0067] The preferred embodiments of the present invention have been specifically described above, but the present invention is not limited to the described embodiments. Those skilled in the art may make various equivalent modifications or substitutions without departing from the spirit of the present invention. These equivalent modifications or substitutions are all included in the scope defined by the claims of this application.

Claims

1. Use of a type I interferon receptor antagonist and / or a type I interferon signaling pathway inhibitor in the preparation of a drug for preventing, treating or alleviating heatstroke.

2. The use according to claim 1, characterized in that The heat stroke includes pre-heat stroke, mild heat stroke and severe heat stroke, as well as diseases and symptoms caused by increased body temperature.

3. The use according to claim 1, characterized in that The type I interferon receptor antagonist is selected from at least one of antibodies, polypeptides and small molecules.

4. The use according to claim 1, characterized in that The type I interferon receptor antagonist is an antibody; and the antibody is selected from at least one of a monoclonal anti-type I interferon receptor antibody and a monoclonal anti-type 1 interferon antibody.

5. The use according to claim 1, characterized in that The type I interferon antagonist includes a type I interferon receptor antibody: InVivoMab anti-mouse IFNAR1 (BE0241).

6. The use according to claim 1, characterized in that The type I interferon antagonist includes an antagonist that targets and blocks the human type I interferon signaling pathway.

7. The use according to claim 6, characterized in that The antagonist that targets and blocks the human type I interferon signaling pathway is selected from at least one of: Anifrolumab, Sifalimumab, and the anti-IFN-α monoclonal antibody Rontalizumab.

8. The use according to claim 1, characterized in that The type I interferon signaling pathway inhibitor includes at least one of the STAT1 inhibitor fludarabine, the JAK1 inhibitor filgotinib, and abrocitinib.

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