Use of baz2b gene in preparation of preparation for regulating viral myocarditis

Abstract

The application provides the technical field of biological medicine, and particularly relates to BAZ2B The application provides application of a gene in preparation of a preparation for regulating viral myocarditis. BAZ2B The gene can be applied in preparation of the preparation for regulating viral myocarditis, and the BAZ2B inhibitor GSK2801 can effectively treat viral myocarditis.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and more particularly to... BAZ2B Application of genes in the preparation of agents that regulate viral myocarditis. Background Technology

[0002] Viral myocarditis (VM) is a common cardiovascular disease in children and young adults, and a significant contributing factor to heart failure in these individuals. The mortality rate of acute VM in young adults is as high as 21%, with a case fatality rate of 45%–55.6%. Besides the direct damage to the myocardium caused by the virus, its pathogenesis is more importantly mediated by immune damage mediated by the combined action of the virus and the body's immune response. The inflammatory cascade caused by excessive activation of immune cells is a key factor in viral infection of cardiomyocytes and its induction of heart failure.

[0003] Currently, the clinical treatment principles for viral myocarditis include symptomatic support, antiviral therapy, and immunomodulatory therapy; there is no specific treatment. Symptomatic support primarily involves diuretics, ACEIs / ARBs, and beta-blockers, which can rapidly relieve heart failure symptoms, reduce cardiac load, and improve cardiac function, but cannot directly target the viral cause. Caution should be exercised when using these medications in patients with acute hypotension or severe heart failure. Antiviral therapy mainly includes interferon and ribavirin, which can inhibit viral replication and are effective against specific viruses during the active viral phase. Type I interferon, due to its highly effective ability to inhibit viral replication, has been studied as a potential treatment for viral myocarditis. However, clinical evidence shows significant side effects, such as fever, bone marrow suppression, or hemolytic anemia, which significantly limits its clinical application, and its efficacy remains highly controversial. Increasing evidence suggests that during the progression of viral myocarditis, myocardial damage is often not dominated by persistent viral replication, but rather mediated primarily by excessive inflammatory and immune responses in the later stages of the disease. In this context, interferon therapy may offer limited benefit and, in some cases, even exacerbate immune-mediated myocardial damage. Furthermore, the use of interferon is often limited in patients with severe heart failure, hemodynamic instability, or those with autoimmune diseases and neuropsychiatric disorders. Immunomodulatory therapies such as glucocorticoids and intravenous immunoglobulin (IVIG) can suppress inflammatory responses and regulate immune-mediated myocardial damage, potentially effective in children or immunocompetent cases, but they also have drawbacks such as promoting viral replication and high costs. In patients with severe or acute cardiogenic shock, mechanical circulatory support (such as IABP or ECMO) can maintain blood flow and organ perfusion, but these methods are life-saving measures and cannot cure viral infection. Therefore, clinical treatment requires a comprehensive approach considering the disease course, viral type, and cardiac function status, employing individualized strategies that combine symptomatic and etiological approaches. However, the aforementioned limitations highlight the urgent need to develop new treatment strategies.

[0004] Following viral myocarditis caused by a viral infection, a large number of inflammatory cells, including macrophages, accumulate at the site of cardiac infection. Macrophages participate in viral clearance primarily through a variety of synergistic effector mechanisms. Upon exposure to pathogens and exogenous stimuli, they produce macrophage extracellular traps (METs). In pathological states, excessive MET production or incomplete degradation can both activate and attract other immune cells by releasing pro-inflammatory cytokines and chemokines, increasing vascular permeability, promoting inflammatory cell infiltration, leading to local tissue damage and exacerbating the inflammatory response. Inhibiting MET formation can alleviate the inflammatory cascade it triggers.

[0005] BAZ2B is a regulatory subunit of the analog switch chromatin remodeling protein (ISWI) family. It participates in chromatin remodeling by binding to the ATPase subunit and plays a crucial role in transcriptional regulation. Transcriptional regulation of histone post-translational modifications plays a vital role in the formation of METs. Summary of the Invention

[0006] The purpose of this invention is to provide BAZ2B Application of genes in the preparation of agents that regulate viral myocarditis.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution: The present invention also provides BAZ2B Application of genes in the preparation of agents that regulate viral myocarditis.

[0008] The present invention also provides the use of BAZ2B inhibitors in the preparation of formulations for the treatment of viral myocarditis.

[0009] Preferably, the BAZ2B inhibitor includes GSK2801.

[0010] Beneficial effects: This inhibitor works by inhibiting... BAZ2B This inhibitor intervenes in gene-related epigenetic regulatory processes, intervening in histone acetylation recognition and transcriptional activation, thereby significantly inhibiting the abnormal generation of macrophage extracellular traps (METs). With restricted MET formation, macrophage overactivation is effectively controlled, the release of pro-inflammatory cytokines and chemokines is significantly reduced, and the recruitment and infiltration of inflammatory cells into myocardial tissue are alleviated, improving vascular permeability and the local inflammatory microenvironment. Based on this, inflammatory damage and structural destruction of myocardial tissue are significantly alleviated, the overall severity of viral myocarditis is reduced, and disease progression is delayed. Therefore, this inhibitor, by targeting the key immunomodulatory link of MET generation, inhibits the amplified inflammatory cascade response, thus playing an important anti-inflammatory and cardioprotective role in CVB3-induced viral myocarditis.

[0011] Under viral infection conditions, the inflammatory microenvironment of myocardial tissue significantly enhances chromatin activation-related modifications, with elevated H3K14ac levels providing an epigenetic basis for the sustained transcription of inflammation-related genes. BAZ2B, a readout protein for H3K14ac modification, is abnormally highly expressed during viral infection and preferentially binds to promoter or enhancer regions enriched with H3K14ac, thereby promoting the transcriptional activation of IRF7. The sustained upregulation of IRF7 further amplifies type I interferon and downstream inflammatory signals, driving excessive macrophage activation and promoting the abnormal formation of macrophage extracellular traps (METs). Excessive formation of METs exacerbates local inflammatory responses and myocardial tissue damage by releasing various pro-inflammatory mediators and toxic proteins, thus driving the progression of viral myocarditis. Conversely, BAZ2B inhibitors, by interfering with their ability to recognize H3K14ac modifications, weaken the transcriptional activity of the IRF7 promoter / enhancer region, inhibit the sustained activation of IRF7, thereby limiting the excessive amplification of interferon signals, reducing abnormal macrophage activation and METs formation levels, and ultimately effectively alleviating myocardial inflammatory response and reducing the severity of viral myocarditis. Attached Figure Description

[0012] Figure 1 The images show the results of myocardial injury in a mouse model of viral myocarditis. A represents the statistics of mouse body weight and survival rate, B represents the echocardiogram, C represents the results of serum biochemical indicators, D represents the results of HE staining, and E represents the results of Masson staining.

[0013] Figure 2 The diagram shows the confirmed results of METs being present in VM and closely related to disease progression. A is the result of immunofluorescence staining, B is the result of immunohistochemical staining, C is the result of detection of expression levels of inflammation and antiviral related genes, and D is the result of Western Blot protein imprinting. Figure 3 The results of screening key host factors to inhibit MET formation are shown in the following diagrams: A shows the qualitative detection results of METs using Sytox Orange nucleic acid dye; B shows the quantitative detection results of METs using the dsDNA assay kit; C shows the MET detection results using indirect immunofluorescence; D shows the screening process of the GeCKO-J774A.1 whole-genome knockout library; and E shows the MET generation results of the knockout cell lines screened by Sytox Orange nucleic acid dye and the MET yield results of the dsDNA assay kit. Figure 4The diagram shows the data preparation of cardiac tissue transcriptome samples in viral myocarditis. A is the flowchart of transcriptome sequencing, B is the statistical graph of mouse body weight and survival rate, C is the echocardiogram, D is the results of HE staining and Masson staining, and E is the results of serum biochemical index detection.

[0014] Figure 5 The diagram shows the verification results of BAZ2B as a key regulatory gene in viral myocarditis. A is the GO and KEGG diagrams, B is the string diagram, C is the differential gene volcano diagram, D is the relative mRNA level diagram, and E is the Western Blot protein imprinting detection results. Figure 6 The results show that BAZ2B deletion inhibits the pathogenesis of viral myocarditis. A shows the genotype identification results of BAZ2B-KO mice in whole-body BAZ2B knockout mice; B shows the genotype identification results of macrophage-specific conditional knockout BAZ2B mice; C shows the experimental flowchart and drug administration regimen; D shows the mouse body weight statistics; E shows the mouse survival rate statistics; F shows the serum biochemical index detection results; G shows the HE staining, Masson staining, and IHC staining results; H shows the detection results of inflammation and antiviral related gene expression levels; and I shows the Western Blot protein imprinting detection results. Figure 7 For the construction of viral myocarditis models and timelines of different dosing regimens; Figure 8 The following graph shows the effect of GSK2801 administration time window on viral myocarditis in mice. In the graph, A is the mouse body weight statistics, B is the serum biochemical index detection results, C is the HE staining and Masson staining results, and E is the IHC staining results. Figure 9 The results of the effects of GSK2801 dose gradient on viral myocarditis in mice are shown in the figure. A is the mouse body weight statistics, B is the serum biochemical index detection results, and C is the HE staining and Masson staining results. Figure 10 The following graphs show the effects of the BAZ2B inhibitor GSK2801 on viral myocarditis in mice. A represents the statistics of mouse body weight and survival rate, B represents the results of serum biochemical indicators, C represents the results of HE staining, D represents the results of IHC staining, E represents the results of expression levels of inflammation and antiviral related genes, F represents the results of Western Blot protein imprinting, and G represents the results of echocardiography. Figure 11 The image shows the results of immunofluorescence detection and transmission electron microscopy detection in Example 5. Detailed Implementation

[0015] The present invention also provides BAZ2BApplication of genes in the preparation of agents that regulate viral myocarditis; The BAZ2B The gene's accession number is Gene ID:407823.

[0016] The present invention also provides the use of BAZ2B inhibitors in the preparation of formulations for the treatment of viral myocarditis.

[0017] In this invention, the BAZ2B inhibitor selectively attenuates the excessive pro-inflammatory response induced by viral infection (TNF-α, IL-1β, IRF7) without significantly affecting antiviral effector factors (IFN-γ) and inflammatory regulatory / systemic response factors (IL-6, IL-10), suggesting that it primarily acts on the macrophage-driven amplification of innate inflammation, rather than overall antiviral immunity. During the development of viral myocarditis (VM), the BAZ2B inhibitor GSK2801 inhibits excessive macrophage activation by suppressing MET activation, preventing macrophage aggregation at the infection site, and ultimately limiting excessive myocardial inflammation and potential heart failure. This provides a new option for the prevention and treatment of viral myocarditis in clinical practice and has significant socioeconomic value.

[0018] In this invention, the BAZ2B inhibitor includes GSK2801; The general structural formula of GSK2801 is as follows: .

[0019] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0020] Example 1 confirms that METs exist in the VM and are closely related to disease progression.

[0021] To clarify whether METs exist in VM and their correlation with disease progression, this invention constructs a mouse model of viral myocarditis. To provide pathological support for exploring the clinical rescue or inhibition of self-tissue damage caused by excessive immune activation, the CVB3 infection dose used in this model was a non-lethal dose.

[0022] Establishment of a viral myocarditis mouse model: Five- to six-week-old male BALB / c mice (purchased from the Experimental Animal Center of Huaxian County, Anyang City, Henan Province, by Scobes Biotechnology Co., Ltd.) were used in the experiment. The mice were housed in the animal facility of the North Campus of Henan University of Medical Sciences (animal ethics approved by the Experimental Animal Ethics Committee of Henan University of Medical Sciences). After three days of acclimatization, a viral myocarditis model was established by intraperitoneal injection of wild-type Coxsackievirus B3 (CVB3) (Nancy strain, purchased from Wuhan Institute of Virology). The viral fluid was diluted to 10⁻⁶ in high-glucose DMEM medium. 5 TCID 50 Each mouse was injected intraperitoneally with 100 μL; control mice were injected with an equal volume of PBS. The daily body weight and survival rate of mice after infection were recorded separately, and the results are as follows: Figure 1 As shown in A; Echocardiography: Echocardiography was performed on mice on days 3, 7, and 14 post-infection to detect left ventricular ejection fraction (LVEF) and fractional shortening (FS). The results are as follows: Figure 1 As shown in B; Serum biochemical marker detection: Blood was collected from mice on days 3, 7, and 14 post-infection, and serum was separated and sent to Xinxiang Yinhai Medical Laboratory Co., Ltd. to detect serum myocardial injury-related biochemical markers (CK, CK-MB, cTnI, AST, LDH, and α-HBDH). The detection instruments used were a Dirui CS-2000 fully automated biochemical analyzer for CK detection via the creatine phosphate substrate method, CK-MB via the immunosuppression method, AST via the aspartate substrate method, LDH via the lactate substrate method, and α-HBDH via the α-ketobutyrate substrate method. cTnI was detected using a MAGLUMI X8 fully automated chemiluminescence immunoassay analyzer via a chemiluminescence method. The results are as follows: Figure 1 As shown in C; HE staining and Masson staining: Mice were sacrificed on days 3, 7, and 14 post-infection. Heart tissue was collected, fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned (4-5 μm). HE and Masson staining were then performed. HE staining was used to observe inflammatory cell infiltration, myocardial fiber arrangement, and necrosis in the myocardial tissue. Masson trichrome staining was used to assess collagen deposition and fibrosis in the myocardial interstitium. Multiple fields of view were randomly selected, and semi-quantitative analysis was performed using image analysis software. The results are as follows: Figure 1 As shown in D and 1E; Myeloperoxidase (MPO) is a specific marker protein for extracellular traps of neutrophils, macrophages, and other myeloid cells (see Sorensen OE, Borregaard N. Neutrophil extracellular traps-the dark side of neutrophils. J Clin Invest. 2016;126(5):1612-20). In this invention, MPO (Recombinant rabbit Anti-Myeloperoxidase antibody; Abcam, AB208670) is used as an indicator protein to perform immunofluorescence staining on heart tissue to indicate macrophage extracellular traps (METs). Paraffin sections were dewaxed, hydrated, and antigen-retentive, and then subjected to immunofluorescence staining and immunohistochemical staining, respectively. Immunofluorescence staining: Cardiac tissue was stained with MPO antibody using immunofluorescence, and imaging was performed using confocal microscopy to analyze the expression and distribution of METs in myocardial tissue and their correlation with macrophage inflammatory responses. Results are as follows: Figure 2 As shown in A; Immunohistochemical staining (IHC staining): Primary antibody against MPO (a macrophage extracellular trap protein) was used for incubation, followed by the addition of a corresponding HRP-labeled secondary antibody; DAB staining was then performed. Results are shown below. Figure 2 As shown in B; Detection of expression levels of inflammation and antiviral related genes: Total RNA was extracted from myocardial tissue and cDNA was synthesized by reverse transcription; real-time quantitative PCR was performed using the SYBR Green method to detect the mRNA expression levels of BAZ2B and inflammation-related genes (IL-1β, IL-6, IL-10, TNF-α, IFN-γ), with GAPDH as an internal reference gene. -ΔΔCt The method was used for relative quantitative analysis, and the results are as follows: Figure 2 As shown in C; Western Blot (WB): Total protein was extracted from myocardial tissue using RIPA lysis buffer, and protein concentration was determined by the BCA method. Equal volumes of protein were subjected to SDS-PAGE electrophoresis and then transferred to a PVDF membrane. The membrane was blocked and incubated with primary and secondary antibodies against inflammation-related proteins, respectively. Chemiluminescence imaging was used to analyze the effects of BAZ2B inhibition or deletion on myocarditis-related signaling pathways (NLRP3, IL-1β, IL-18, caspase-8, MPO). Results are as follows: Figure 2 As shown in D; Analysis of mouse weight change curves and survival rates at different time points (3d, 7d, 14d) after CVB3 infection showed that the weight loss of mice was most significant at 7d post-infection, indicating that the degree of myocardial damage was the most severe at this time point. Quantitative analysis of echocardiographic results in mice showed that, compared with the control group, the viral myocarditis group experienced the most significant decline in cardiac systolic function 7 days after infection, with a partial recovery trend after 14 days. Serum biochemical indicators related to myocardial injury showed that the indicators increased significantly 7 days after infection, suggesting that myocardial cell damage was most severe at this time point. Compared with the control group, HE staining of cardiac tissue showed mild inflammatory cell infiltration 3 days after CVB3 infection, a large number of inflammatory cells and disordered myocardial fiber structure were observed in myocardial tissue at 7 days, and the degree of inflammation was relieved at 14 days. Masson staining of cardiac tissue showed a significant increase in myocardial interstitial collagen deposition 7 days after infection, suggesting that inflammation-related myocardial fibrosis was most pronounced at this time point. Immunofluorescence staining results showed that, compared with the control group, the number of F4 / 80 positive cells in myocardial tissue increased significantly with the extension of CVB3 infection time, and they were clearly co-localized with MPO signals. A large amount of yellow fluorescence was observed in the Merge image, indicating that the formation of METs in myocardial tissue in viral myocarditis reached its peak at 7 days after infection; the immunohistochemical staining results were consistent with the immunofluorescence trend. The results of detecting the mRNA expression levels of inflammatory factors in myocardial tissue of each group showed that the expression levels of inflammatory factors increased significantly 7 days after infection, and the inflammatory response was most active. Immunoblotting results of myocardial inflammation-related proteins and METs-related proteins showed that, compared with the control group and other time points, the expression levels of NLRP3, IL-1β, IL-18 and MPO proteins in myocardial tissue were significantly increased 7 days after infection, suggesting that inflammasome activation and METs formation were most significant at this time point. The above results confirm that METs exist in VM and are closely related to disease progression. METs and inflammatory responses are most significant at 7 days after infection. Therefore, this infection dose and detection time point were selected for subsequent experiments.

[0023] Example 2 confirms that BAZ2B is a key regulatory gene in viral myocarditis.

[0024] To screen key genes regulating MET formation from a genome-wide perspective, this invention initially constructed in vitro... Figure 3The mouse macrophage J774A.1-METs cell model shown in AC (referencing the master's thesis from Huazhong Agricultural University, "Screening of C5a-related genes affecting METs formation using a mouse whole-genome CRISPR / Cas9 knockout library," DOI: 10.27158 / d.cnki.ghznu.2022.000919) was used. The mouse macrophage line J774A.1 was stimulated with 10 μg / mL of the complement system immunomodulator C5a, and Sytox Orange nucleic acid dye was added. The release of extracellular DNA from the macrophages was observed using a fluorescence microscope. Figure 3 As shown in A, filamentous material was visible in the extracellular space 2 hours after stimulation. The DNA yield in the extracellular supernatant, as quantitatively detected by the dsDNA assay kit, was significantly different from that in the control group. Figure 3 B). To confirm whether the extracellular material formed by C5a stimulation of J774A.1 cells is METs, an indirect immunofluorescence assay was performed on the extracellular structure using an anti-Histone H3 specific antibody. A significant Histone H3-DNA complex was detected in the cells, indicating that the substance produced is a macrophage extracellular trap carrying histones. Figure 3 C). A mouse whole-genome knockout library, J774A.1-GeCKO, was constructed using CRISPR / Cas9 technology to screen for key host factors that inhibit MET formation. For example... Figure 3 D used the complement component C5a protein to stimulate the genome-wide knockout cell line J774A.1-GeCKO. Since C5a stimulation led to the death of J774A.1 cells, but cells with knocked-out genes inhibiting MET formation survived after C5a stimulation, the surviving cells after C5a stimulation of the library were collected. High-throughput sequencing of the sgRNA sequences in the surviving cells' genomes was performed to identify potential genes related to inhibiting MET formation and macrophage death. Functional validation of knockout and complement candidate genes was then conducted in new J774A.1 cells. Figure 3 (E) It was found that BAZ2B deficiency had a significant inhibitory effect on MET formation, indicating that the epigenetic factor BAZ2B is a potential regulator of MET generation.

[0025] Based on the results of in vitro experiments, to verify whether BAZ2B works in an in vivo mouse model of viral myocarditis, 5-week-old male BALB / c mice were selected in this invention. 5 TCID 50 As the infection dose, heart tissue samples from control and model mice were sent to Shanghai Paisenno Biotechnology Co., Ltd. for transcriptome sequencing. Figure 4 A), and the mouse weight change curve and survival rate were recorded simultaneously. Figure 4B), cardiac ultrasound was performed on mice on day 7 after infection. Figure 4 C), according to the method described in Example 1, and HE staining of the heart tissue of two groups of mice ( Figure 4 D) Masson staining ( Figure 4 D) and detection of serum myocardial injury-related biochemical indicators (CK, CK-MB, cTnI) Figure 4 E), HE staining showed a significant increase in inflammatory cell infiltration in the myocardial tissue of the CVB3-infected group; Masson staining showed a significant increase in collagen deposition in the myocardial interstitium, suggesting aggravated myocardial fibrosis; the above results and serum test results indicate that the viral myocarditis mouse model was successfully established; Differential expression analysis was performed on transcriptome data. The screening criteria were |log2FC|≥1 and P<0.05. The differentially expressed genes were analyzed for GO functional enrichment and KEGG pathway using the Gene Cloud online software of Shanghai Paisenuo Biotechnology Co., Ltd. The hypergeometric test was used and multiple corrections were performed using the Benjamini-Hochberg method. After adjustment, a P value <0.05 was considered statistically significant. GO analysis results ( Figure 5 A) indicates that viral myocarditis elicits significant intracellular inflammation and changes in the innate immune response in cardiac cells, with cell membrane alterations involved in immune activation and mediator release; KEGG chord diagram ( Figure 5 B) The results showed that many differentially expressed genes were involved in oxidative phosphorylation, the PI3K-Akt signaling pathway, apoptosis, TNF, and MAPK signaling pathways, indicating that innate immune processes play a key role in maintaining intracellular homeostasis and responding to viral invasion. Among the significantly differentially expressed genes, molecules related to inflammatory pathways were screened. Volcano plot results showed that the genes IRF7, MYH7, GZMA, and BAZ2B were significantly upregulated in the heart tissue of VMC group mice. Figure 5 C), subsequent qPCR ( Figure 5 D) and Western Blot ( Figure 5 E) Further validation of the transcriptome sequencing analysis results showed that the key METs indicator protein MPO was highly expressed in VM, suggesting that the BAZ2B gene not only plays a significant role in the generation of METs, but may also play a regulatory role in VM. To further clarify whether BAZ2B regulates MET formation in vivo, we commissioned Shanghai Southern Model Biotechnology Co., Ltd. to construct a BAZ2B systemic knockout mouse system. - / + and macrophage-specific conditional knockout BAZ2B mice BAZ2B Flox- / +The specific construction principle involves using CRISPR / Cas9 technology to introduce mutations through non-homologous recombination repair, causing a frameshift and loss of function in the BAZ2B gene protein, thus obtaining BAZ2B systemic knockout heterozygous mice. - / + The specific procedure involves designing sgRNA targeting the key exon of BAZ2B, injecting it into fertilized eggs, and selecting heterozygous mice with the BAZ2B gene deletion (catalog number: NM-KO-2110334). After mating and breeding in the SPF mouse house, genotyping was performed to obtain homozygous mice named BAZ2B-KO. In addition, using the principle of homologous recombination, the BAZ2B gene was modified with flux through homologous recombination in fertilized eggs to obtain BAZ2B gene conditional knockout heterozygous mice C57BL / 6J-Baz2b. em1(flox)Cya (Catalogue No.: NM-CKO-220456); and Cx3cr1 Cre Through mating and breeding with tool mice, macrophage-specific conditional knockout BAZ2B mice were eventually obtained. Flox + / + The genotype was confirmed by tail genotyping to obtain homozygotes named BAZ2B-Flox (wild-type littermates were used as the control group). The results of mouse genotyping analysis are shown in the figure. Figure 6 AB); Mice with viral myocarditis were constructed as the virus group (VM) according to the method described in Example 1. Existing studies have shown that METs are composed of macrophage chromosomal DNA and various particulate proteins such as MPO and histones. Treatment with DNase I can degrade chromosomal DNA. Therefore, starting on the 3rd day after viral infection, mice were intraperitoneally injected with DNase I at a single injection of 100 μg / day / mouse, once a day for a total of five injections (References: 10.3390 / jcm11154349; 10.1002 / 1878-0261.12787), which served as the METs treatment group control (VM+DNase I). The viral myocarditis model was CVB3. The virus was caused by infection of the mouse heart. Therefore, 10 MIU / kg / mouse of interferon IFN-β was injected intraperitoneally 6 hours after infection, starting from day 0 of infection, once every other day until day 7, for a total of 4 injections (reference: 10.1152 / ajpheart.00154.2007). This group was set as the virus treatment control (VM+IFN-β). The negative control group was treated with the same volume of PBS injected intraperitoneally into normal mice (SHAM). The experimental flowchart is shown in 6C. The body weight change curves of mice in the BAZ2B-KO, BAZ2B-Flox, VM, VM+DNaseI, VM+IFN-β, and SHAM groups were recorded. Figure 6 D) and survival rate were recorded. Figure 6E), on day 7 after infection, CK-MB levels were measured in the heart tissues of both groups of mice according to the method described in Example 1. Figure 6 F), HE staining, Masson staining, immunohistochemical staining ( Figure 6 G); During the development of viral myocarditis, a large number of mononuclear macrophages accumulate and activate in the heart, NF-κB inflammatory pathway and apoptosis are activated, and a large number of inflammatory factors are released, leading to impaired cardiac function. To detect the effect of BAZ2B intervention on the production of METs on the production of inflammatory factors, the expression levels of inflammatory factors TNF-α, IL-1β, IL-6, IFN-γ, and IL-10 were detected according to the method described in Example 1. Figure 6 H); Western blotting was used to detect the expression of inflammation-related proteins IL-1β, IL-6, NLRP3, and METs phenotypic marker protein MPO, etc. Figure 6 I); The above experimental results consistently show that BAZ2B deficiency significantly inhibits METs production and alleviates the severity of viral myocarditis by reducing the release of inflammatory factors.

[0026] Example 3: Effects of GSK2801 Dosing Time Window and Dosage Gradient on Viral Myocarditis in Mice

[0027] Based on the confirmation that BAZ2B participates in regulating MET formation and affects inflammatory damage in viral myocarditis, this invention systematically evaluates the optimal intervention strategy for the BAZ2B inhibitor GSK2801 (Selleck, S7231) in a viral myocarditis model, focusing on both the dosing time window and dose gradient. By applying the inhibitor GSK2801 in the disease model and optimizing the dosing sequence and dosage, this invention constructs a systematic pharmacodynamic evaluation system, thereby demonstrating the application potential of BAZ2B inhibition in improving myocardial injury and inflammatory response.

[0028] Dosing time window: Construction of viral myocarditis model and timeline of different drug administration regimens, as shown in the figure. Figure 7As shown; a viral myocarditis model was constructed as the CVB3 infection group (VM) according to the method described in Example 1, and the same volume of PBS was injected as the control group (SHAM); IFN-β intervention group (VM+IFN-β): IFN-β (10 MIU / kg) was administered intraperitoneally every other day starting 6 hours after CVB3 infection and continued until the end of day 7, for a total of 4 injections; GSK2801 intervention group (VM+GSK2801-post 3days): GSK2801 (30 mg / kg) was continuously administered by gavage starting on day 3 after CVB3 infection, once a day until the end of the experiment on day 7, for a total of 5 times (Reference: 10.1021 / acs.jmedchem.5b00209); GSK2801 combined with IFN-β intervention group (VM+IFN-β+GSK2801): IFN-β was administered in the same manner as above, and GSK2801 was continuously administered by gavage starting on day 0 after CVB3 infection until day 4, for a total of 5 times.

[0029] Record the body weight of mice in each group ( Figure 8 A), echocardiography was performed on mice in each group. Figure 8 B) CK-MB level detection ( Figure 8 C); Heart tissue was collected from mice on day 7 after CVB3 infection, and the heart tissue was stained with hematoxylin and eosin (HE). Figure 8 D) Masson staining ( Figure 8 D) and immunohistochemical staining ( Figure 8 E); Overall, the experimental results showed that CVB3 infection can lead to significant weight loss, impaired cardiac function, and inflammatory damage to myocardial tissue, while drug intervention can alleviate these pathological changes to varying degrees. Compared with the infection group, GSK2801 intervention 3-7 days after infection significantly reduced the trend of weight loss in mice. The GSK2801 combined with IFN-β intervention group, due to early intervention with GSK2801, actually had a lower weight compared to the acute phase intervention group (GSK2801 intervention group). Combined with experimental result 1, which showed that METs were already produced 3 days after infection, it suggests that early intervention with GSK2801 may hinder the implementation of METs' immune killing effect. Echocardiography showed that CVB3 infection significantly reduced left ventricular ejection fraction and fractional shortening, indicating impaired cardiac function. GSK2801 treatment significantly improved these indicators, showing a protective trend comparable to or better than IFN-β. Serological tests further showed that CVB3 infection induced elevated levels of the myocardial injury marker CK-MB, while GSK2801 intervention effectively reduced this marker. Histological analysis was consistent with the functional results described above. HE staining showed that CVB3 infection led to significant inflammatory cell infiltration and myocardial structural damage, while GSK2801 treatment significantly reduced inflammatory infiltration and improved the integrity of myocardial fiber arrangement. Masson staining results indicated increased collagen deposition in the myocardial interstitium of the infected group, while drug intervention significantly inhibited collagen deposition and fibrosis. Immunohistochemical detection showed a significant increase in MPO-positive inflammatory cells in the myocardial tissue after infection, while GSK2801 treatment significantly reduced their infiltration level, further supporting its anti-inflammatory protective effect; suggesting that drug intervention within this time window has a relatively ideal protective effect.

[0030] Dose gradient: To better implement its clinical application, an experiment was further conducted to determine the minimum effective dosage of GSK2801. The dose gradients of 7.5 mg / kg, 15 mg / kg, and 30 mg / kg were compared: the body weight of mice in each group was recorded. Figure 9 A), CK-MB levels were measured in each group of mice. Figure 9 B); Mouse heart tissue was collected on day 7 after CVB3 infection, and the heart tissue was stained with HE and Masson staining (B). Figure 9 C); Analysis of comprehensive weight changes, serum CK-MB levels, and HE and Masson staining results of myocardial tissue revealed that all doses exhibited varying degrees of cardioprotective effects. Specifically, 7.5 mg / kg significantly reduced infection-induced weight loss, decreased myocardial injury indicators, and improved inflammatory infiltration and collagen deposition, suggesting that this dose could achieve a relatively effective intervention. Overall, the results indicate that GSK2801, when initiated during the inflammatory development phase after infection and administered at a relatively low dose, can produce significant protective effects, providing a reasonable basis for subsequent mechanism studies and potential clinical translation.

[0031] Example 4: Effect of the BAZ2B inhibitor GSK2801 on viral myocarditis in mice.

[0032] Based on the optimized dosing parameters obtained in the aforementioned embodiments, this implementation case sets up multiple treatment control regimens under these conditions to further verify the efficacy and mechanism of the BAZ2B inhibitor GSK2801 in a viral myocarditis model.

[0033] Referring to the method described in Example 3, the following groups were set up: CVB3 infection group (VM), control group (SHAM), IFN-β intervention group (+IFN-β), GSK2801 intervention group (+GSK2801), and DNase I intervention group (+DNase I); wherein the gavage dose of GSK2801 in the GSK2801 intervention group was 7.5 mg / kg; the administration methods of IFN-β and DNase I were as described above.

[0034] The daily body weight and survival rate of mice after infection were recorded separately. Figure 10 A); Echocardiography was performed on mice in each group on day 7 after infection. Figure 10 G) Serum biochemical marker detection (CK, CK-MB, AST) detection ( Figure 10 B) Masson staining ( Figure 10 C) Immunohistochemical staining ( Figure 10 D) Detection of inflammation and antiviral related gene expression levels (TNF-α, IL-1β, IFN-γ, IL-6, IL-10) Figure 10 E) and WB detection ( Figure 10 F); The results showed that in the BALB / c mouse model of viral myocarditis, GSK2801 treatment could alleviate weight loss and improve survival in the model animals, exhibiting a similar trend to the IFN-β intervention group. Serum biochemical markers and histological analysis consistently showed that GSK2801 intervention reduced inflammatory cell infiltration in myocardial tissue and improved the integrity of myofibrous structure. Masson staining indicated decreased collagen deposition in the myocardial interstitium, suggesting an inhibitory effect on inflammation-related fibrosis. Simultaneously, the decrease in MPO immunohistochemical positivity indicated that METs production was inhibited. Further analysis of inflammation-related molecular levels revealed that the expression of inflammation- and antiviral-related genes such as IL-1β, IFN-γ, IL-6, and IL-10 was upregulated in the viral myocarditis model, but decreased to varying degrees after GSK2801 intervention. In the mid-to-late stages of infection, the inflammatory factors IFN-γ and IL-6 showed no significant difference between the GSK2801-treated group and the viral infection group, further suggesting that they mainly act on the macrophage-driven amplification of innate inflammation, rather than overall antiviral immunity. Western blot results also showed that the expression of related signaling pathway proteins was regulated. In terms of functional assessment, echocardiographic results indicated that GSK2801 improved left ventricular ejection fraction and fractional shortening, with an overall protective effect comparable to the IFN-β-treated group and showing a similar cardioprotective trend to the DNase I-treated group.

[0035] Example 5: Effects of the BAZ2B inhibitor GSK2801 on MET formation and cardiomyocyte ultrastructural damage in viral myocarditis

[0036] To observe the effects of the BAZ2B inhibitor GSK2801 on the formation of macrophage extracellular traps (METs) and ultrastructural damage of cardiomyocytes in viral myocarditis, myocardial tissues from the CVB3 infection group (VM), control group (SHAM), and GSK2801 intervention group (+GSK2801) in Example 4 were subjected to immunofluorescence detection and transmission electron microscopy (TEM) examination. The results are as follows: Figure 11 As shown; TEM detection method: Fresh myocardial tissue was cut into 1mm pieces 3 Small fragments were fixed with electron microscopy solution, then subjected to gradient dehydration, embedding, and ultrathin sectioning. Transmission electron microscopy was used to observe the mitochondrial structure, myofibril arrangement, and organelle damage of cardiomyocytes to assess the effect of BAZ2B regulation on macrophage morphology and myocardial ultrastructural damage.

[0037] Figure 11 The image above is a schematic diagram of the immunofluorescence colocalization results of cardiac tissue in viral myocarditis. DAPI labels the cell nuclei, F4 / 80 labels the infiltrating macrophages, and MPO is a specific marker protein for METs. The results show that in the viral myocarditis model, MPO and F4 / 80 signals are significantly enhanced and highly colocalized, suggesting that macrophage METs are formed in large quantities in myocardial tissue.

[0038] Figure 11 The image below shows the electron microscopy results. In the image, N represents the macrophage nucleus; ★ represents the mitochondria; and ◆ represents the lysosomes within the dashed circle on the left. The symbols represent phagosomes / phagolysosomes; # represents autophagosomes / autolysosomes; and the arrow represents METs (Macrophage Extracellular Traps). Based on immunofluorescence confirmation of the presence of extracellular chromatin in macrophages, TEM showed that macrophages were chemotactically attracted to the intermyocardial space after viral infection of mouse myocardial tissue. Furthermore, the chromatin in the macrophage nucleus underwent significant decondensation accompanied by focal nuclear structural disruption, providing ultrastructural support for the chromatin release process. Combined with indirect immunofluorescence results, extracellular chromatin signals were visible around macrophages: significant decondensation of chromatin in the macrophage nucleus, decreased nuclear electron density, local discontinuities in the nuclear membrane structure, and some chromatin-like material extending into the cytoplasm and extracellular space, suggesting the occurrence of chromatin release.

[0039] Figure 11Image B on the right shows the transmission electron microscopy (TEM) results of myocardial tissue from different groups. In each group, the control group showed regular arrangement of myofibrils and intact mitochondrial structure in every two fields of view. In the viral myocarditis model group, enlarged intercellular spaces, mitochondrial swelling, and cristae destruction were observed. Macrophages were present in the intercellular spaces and around capillaries, accompanied by a large amount of extracellular reticular structure deposition, indicating significant METs-related ultrastructure. After treatment with GSK2801, the overall morphology of myocardial mitochondria and myofibril structure was significantly improved, and the extracellular reticular structure of macrophages was significantly reduced. This indicates that the inhibitor can inhibit METs formation and reduce myocardial cell ultrastructural damage.

[0040] As can be seen from the above embodiments, the present invention provides BAZ2B The application of genes in the preparation of agents that regulate viral myocarditis: BAZ2B inhibitor GSK2801 can effectively treat viral myocarditis.

[0041] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. BAZ2B Application of genes in the preparation of agents that regulate viral myocarditis.

2. Application of BAZ2B inhibitors in the preparation of formulations for the treatment of viral myocarditis.

3. The application according to claim 2, characterized in that, The BAZ2B inhibitor includes GSK2801.

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