Application of BMAL1 activator ISX-9 in preparation of medicine for preventing and treating thoracic aortic aneurysm and dissection

Abstract

The invention discloses an application of a BMAL1 activator ISX-9 in preparation of a medicine for preventing and treating thoracic aortic aneurysm and dissection. The invention provides an application of a BMAL1 activator ISX-9 in preparation of a medicine for preventing and / or treating thoracic aortic aneurysm and dissection. The step of preventing and treating the TAAD comprises the substeps of reducing the incidence rate of the TAAD and relieving cavities and / or fractures of the middle-layer structure of the thoracic aorta. According to the application disclosed by the invention, ISX-9 is subjected to intraperitoneal injection in a TAAD mouse model, and a result shows that the ISX-9 can be used for remarkably reducing BAPN-induced mouse thoracic aorta VSMC apoptosis and elastic fiber degradation and improving the histopathologic change of TAAD; the blood pressure, the body weight and the blood fat level of the mouse are not obviously influenced by the administration of the ISX-9, and the ISX-9 has good safety, so that the ISX-9 provided by the invention has a good clinical application prospect.

Description

TECHNICAL FIELD

[0001] The application relates to an application of a BMAL1 activator ISX-9 in preparation of a drug for preventing and treating thoracic aortic aneurysm and dissection, and belongs to the technical field of biological medicine. BACKGROUND

[0002] Thoracic aortic aneurysm and dissection (TAAD) is a kind of fatal cardiovascular disease, and the main pathological features are abnormal expansion of the aortic wall (aneurysmal lesions) or intimal tear to form a false lumen (dissection). The core pathological mechanism is vascular smooth muscle cell (VSMC) apoptosis, elastic fiber degradation and medial degeneration, which finally leads to a significant increase in the risk of aortic rupture. TAAD has an insidious onset, but the mortality rate is as high as 50% when it acutely attacks, which seriously threatens the life of the patient. At present, the clinical treatment mainly adopts open surgery or endovascular repair, but the surgery has great trauma and many complications, and cannot reverse the early lesions. Although some drugs (such as beta blockers) can delay the disease, there is no targeted treatment method that can effectively inhibit VSMC apoptosis or block the disease progression. Therefore, it is urgent to develop a drug targeting the core pathological mechanism of TAAD.

[0003] Studies have shown that VSMC apoptosis is the key mechanism leading to VSMC loss in the pathogenesis of TAAD. VSMC apoptosis is a programmed cell death induced by internal and external factors, and is mainly triggered by mitochondrial pathway, death receptor pathway and mechanical stress. Regulating VSMC apoptosis may be a potential method for preventing and treating TAAD. ISX-9 is proved to be a highly efficient BMAL1 activator through drug screening, and can play a dual role of promoting BMAL1 transcription and translation and activating BMAL1 activity. However, it is not clear whether ISX-9 can regulate VSMC apoptosis and reduce the incidence of TAAD. Therefore, it is of great value to study whether ISX-9 can affect the incidence of TAAD by inhibiting VSMC apoptosis. SUMMARY

[0004] The purpose of the present application is: in order to overcome the deficiencies of the above-mentioned prior art, the purpose of the present application is to provide an application of a BMAL1 activator ISX-9 in preparation of a drug for preventing and treating thoracic aortic aneurysm and dissection. The present application researches and finds that the BMAL1 activator ISX-9 can inhibit VSMC apoptosis, and achieve prevention and treatment of TAAD.

[0005] In order to achieve the above-mentioned purpose, the present application provides an application of a BMAL1 activator ISX-9 in preparation of a drug for preventing and / or treating thoracic aortic aneurysm and dissection.

[0006] The prevention and treatment of TAAD include reducing the incidence of TAAD, reducing the dissection and / or rupture of the tunica media of the thoracic aorta.

[0007] Preferably, the medicine comprises an effective component and a pharmaceutically acceptable adjuvant or carrier, and the effective component comprises ISX-9.

[0008] In some embodiments of the present application, the medicine takes ISX-9 as the only effective component.

[0009] Preferably, the dosage form of the medicine comprises a suspension, a tablet, a granule, a capsule, an injection, a powder, a dripping pill, a suppository, an aerosol or a drop.

[0010] Preferably, the administration route of the medicine comprises intraperitoneal injection, intravenous injection, intramuscular injection, subcutaneous injection, oral administration, sublingual administration, nasal administration or atomization administration.

[0011] Compared with the prior art, the present application has the following beneficial effects: (1) The present application finds that the deficiency of the core biological rhythm gene BMAL1 can significantly increase the incidence of TAAD in mice by promoting VSMC apoptosis through bioinformatics analysis; ISX-9 is a highly efficient BMAL1 activator, which can play a dual role of promoting the transcription and translation of BMAL1 and activating the activity of BMAL1; ISX-9 can significantly increase the BMAL1 protein level of the tunica media of the thoracic aorta.

[0012] (2) The present application induces a mouse TAAD model by continuously feeding the mice with 0.25% BAPN for 28 days, and intraperitoneal injection of ISX-9 can significantly reduce the VSMC apoptosis and elastic fiber degradation of the thoracic aorta of the BAPN-induced mice, and improve the histopathological changes of TAAD; and the administration of ISX-9 does not significantly affect the blood pressure, body weight and blood lipid level of the mice, and has good safety, so the ISX-9 provided by the present application has good clinical application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0013] Figure 1Figure 8 is a comparison of the expression levels of BMAL1 in the thoracic aorta of the control group and the TAAD group; wherein A is a KEGG enrichment analysis chart of the differentially expressed genes in the thoracic aorta tissue of the control group and the TAAD group; B is a GSEA enrichment analysis chart of the differentially expressed genes in the thoracic aorta tissue of the control group and the TAAD group; C is a comparison of the mRNA levels of BMAL1 in the thoracic aorta tissue of the control group and the TAAD group in the center; D is a comparison of the mRNA levels of BMAL1 in the thoracic aorta tissue of the control group and the TAAD group in the public data set GSE140947; E is BMAL1 immunofluorescence staining of the thoracic aorta tissue of the control group and the TAAD group; F is statistical analysis of the corresponding BMAL1 immunofluorescence staining; G is BMAL1 Western blot detection of the thoracic aorta tissue of the control group and the TAAD group; H is a schematic diagram of the TAAD modeling of mice; I is BMAL1 immunofluorescence staining of the thoracic aorta tissue of the control group and the TAAD group of mice; J is statistical analysis of the corresponding BMAL1 immunofluorescence staining; K is BMAL1 Western blot detection of the thoracic aorta tissue of the control group and the TAAD group of mice; 3PM in E-G represents a sample at 3 pm, and 9PM represents a sample at 9 pm.

[0014] Figure 2 Figure 9 is the effect of BMAL1 knockdown on the incidence of TAAD in mice; wherein A is a schematic diagram of the TAAD modeling of mice with whole-body knockdown of BMAL1; B is a survival curve of mice with whole-body knockdown of BMAL1 in the TAAD modeling state; C is a typical gross anatomy picture; D is a TAAD incidence statistic of mice with whole-body knockdown of BMAL1; E is a representative picture of hematoxylin-eosin and elastic fiber staining of mice with whole-body knockdown of BMAL1; F is a thoracic aorta diameter statistic; G is an elastic fiber degradation index statistic; H is a co-localization staining of BMAL1 and VSMC in the thoracic aorta of humans; I is a co-localization staining of BMAL1 and VSMC in the thoracic aorta of mice; J is a schematic diagram of the TAAD modeling of mice with VSMC-specific knockdown of BMAL1; K is a survival curve of mice with VSMC-specific knockdown of BMAL1 in the TAAD modeling state; L is a typical gross anatomy picture; M is a TAAD incidence statistic of mice with VSMC-specific knockdown of BMAL1; N is a representative picture of hematoxylin-eosin and elastic fiber staining of mice with VSMC-specific knockdown of BMAL1; O is a thoracic aorta diameter statistic; P is an elastic fiber degradation index statistic.

[0015] Figure 3Figure 9. Effects of BMAL1 deficiency on VSMC apoptosis; A, volcano plot of differentially expressed genes from the mRNA transcriptome of thoracic aorta from BMAL1 knockdown mice; B, results of Gene Ontology (GO) enrichment analysis; C, results of GSEA enrichment analysis; D, spatial transcriptome annotation plot of thoracic aorta from BMAL1 knockdown mice; E, quantification of spatial transcriptome of Caspase 3, 7 and 8; F, differences in TUNEL and Cleaved caspase-3 levels in wild type and BMAL1 knockdown mice; G, quantitative statistical analysis of TUNEL and Cleaved caspase-3 staining; H, differences in BCL-2 levels in wild type and BMAL1 knockdown mice; I, effects of BMAL1 knockdown and overexpression on BCL-2 levels in VSMC cultured in vitro; J, quantitative statistics of BMAL1 levels; K, quantitative statistics of BCL-2 levels; L, effects of BMAL1 knockdown on cell viability of VSMC cultured in vitro; M, effects of BMAL1 knockdown on apoptosis signal TUNEL of VSMC cultured in vitro; N, quantitative statistics of the proportion of TUNEL positive VSMC.

[0016] Figure 4 Figure 10. Effects of ISX-9 on increasing aortic BMAL1 levels; A, effects of ISX-9 on BMAL1 transcriptional levels in thoracic aorta of mice; B, effects of ISX-9 on BMAL1 protein levels in thoracic aorta of mice; C, quantitative statistics of immunostaining.

[0017] Figure 5Figure 9 shows that ISX-9 reduces the incidence of TAAD in BMAL1 knockdown mice; wherein, A is a schematic diagram of ISX-9 prevention of TAAD in BMAL1 knockdown mice; B is the survival curve of BMAL1 knockdown mice after prophylactic administration of ISX-9; C is the incidence of TAAD in BMAL1 knockdown mice after administration of ISX-9; D is GSEA enrichment analysis of transcriptomics sequencing; E is a heat map of apoptosis-related differential genes of transcriptomics sequencing; F is GO enrichment analysis of ISX-9 up-regulated genes; G is GO enrichment analysis of ISX-9 down-regulated genes; H is immunofluorescence staining of TUNEL and c-MYC levels in the aorta of BMAL1 knockdown mice after administration of ISX-9; I is a statistical chart of immunofluorescence staining; J is western blotting of c-MYC and BCL-2 levels in the aorta of BMAL1 knockdown mice after administration of ISX-9; K is a schematic diagram of ISX-9 prevention of TAAD in wild-type mice; L is the survival curve of wild-type mice after prophylactic administration of ISX-9; M is the incidence of TAAD in wild-type mice after prophylactic administration of ISX-9; N is GSEA enrichment analysis of transcriptomics sequencing; O is a heat map of apoptosis-related differential genes of transcriptomics sequencing; P is a schematic diagram of ISX-9 treatment of TAAD in BMAL1 knockdown mice; Q is the survival curve of BMAL1 knockdown mice after therapeutic administration of ISX-9; R is the incidence of TAAD in BMAL1 knockdown mice after therapeutic administration of ISX-9; S is a schematic diagram of ISX-9 treatment of TAAD in wild-type mice; T is the survival curve of wild-type mice after therapeutic administration of ISX-9; U is the incidence of TAAD in wild-type mice after therapeutic administration of ISX-9.

[0018] Figure 6 Figure 10 shows that ISX-9 has little effect on the body weight, blood pressure and blood lipid levels of mice; wherein, A is the body weight change curve of mice in the solvent control group and the ISX-9 administration group under the condition of BAPN modeling; B is a comparison of systolic blood pressure between the two groups; C is a comparison of diastolic blood pressure between the two groups; D is a comparison of serum triglyceride levels between the two groups; E is a comparison of serum total cholesterol between the two groups; F is a comparison of low-density lipoprotein cholesterol levels between the two groups; G is a comparison of serum high-density lipoprotein cholesterol levels between the two groups. DETAILED DESCRIPTION

[0019] In order to make the present application more obvious and easy to understand, the preferred embodiments are described in detail below, with reference to the accompanying drawings.

[0020] The test methods or test methods described in the following examples are all conventional methods unless otherwise specified; the reagents and materials are obtained from conventional commercial channels or prepared by conventional methods unless otherwise specified.

[0021] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0022] The ISX-9 described in this invention has the CAS No. 832115-62-5 and the molecular formula: C 11 H 10 N2O2S (available commercially).

[0023] Example (1) BMAL1 levels decreased significantly in clinical and mouse studies of TAAD. Thyral aortic tissues from 10 control groups and 10 TAAD groups were collected for mRNA transcriptomics sequencing. KEGG enrichment analysis showed that differentially expressed genes were significantly enriched in circadian rhythm-related pathways. Figure 1 A). GSEA enrichment analysis yielded similar conclusions ( Figure 1 B). Data from our center (Department of Cardiac Surgery, Zhongshan Hospital Affiliated to Fudan University) and public databases both indicate that BMAL1 mRNA levels were significantly decreased in the TAAD group. Figure 1 C-1D). Immunofluorescence and Western blot assays confirmed the decrease of BMAL1 in the TAAD aorta at the protein level. Figure 1 E-1G). We established a mouse TAAD model using a 0.25% β-aminopropionitrile (BAPN) feeding regimen. Figure 1 H). Similarly, both immunofluorescence and Western blotting experiments showed that the protein level of BMAL1 was significantly decreased in the thoracic aorta of TAAD mice. Figure 1 I-1K).

[0024] (2) BMAL1 deficiency significantly increases the incidence of TAAD in mice. This invention uses C57BL / 6 mice housed in an SPF-grade animal facility under a 12-hour alternating light and dark environment at a constant temperature of 23°C. Bmal1 heterozygous knockout (BKD) mice were obtained by crossbreeding Bmal1-flox mice with E2a-Cre mice. Thirty wild-type (WT) and BKD mice from the same batch were randomly selected and fed a diet containing 0.25% BAPN for 4 weeks at 4 weeks of age to establish a TAAD model. Figure 2 A). During the modeling period, 50% of wild-type mice died from thoracic aortic rupture, and 7% developed thoracic aortic aneurysms, although they had not yet dissected or ruptured. In contrast, 86% of BKD mice died from thoracic aortic rupture, and 7% developed unruptured thoracic aortic aneurysms. Figure 2 B-2D). Morphological staining and statistical results showed that the diameter of the thoracic aorta and the degree of elastic fiber degradation in BKD mice were significantly higher than those in the WT group, pathologically confirming the impact of BMAL1 deficiency on the pathogenesis of TAAD. Figure 2E-2G). Immunofluorescence experiments showed that BMAL1 protein levels were significantly decreased in VSMCs in human and mouse TAAD aortas Figure 2 H-2I). Therefore, we generated VSMC-specific BMAL1 knockdown mice by crossing Acta2 cre and Bmal1 flox / flox Further, we generated VSMC-specific BMAL1 knockdown mice by crossing Acta2 cre+ Bmal1 flox / + Similarly, the incidence of TAAD was significantly increased in this line of mice Figure 2 J-2P).

[0025] (3) BMAL1 deficiency promotes VSMC apoptosis To further investigate the mechanism of BMAL1 deficiency increasing TAAD, we collected thoracic aortas from wild-type and BMAL1 knockdown mice for mRNA level transcriptome sequencing, which showed that BMAL1 deficiency significantly activated the apoptosis-related pathway Figure 3 A-3C). Spatial transcriptome sequencing Figure 3 D-3E), histological staining Figure 3 F-3G) and Western blotting Figure 3 H) further demonstrated that BMAL1 deficiency promoted VSMC apoptosis. We extracted human aortic primary VSMCs to further demonstrate the link between BMAL1 deficiency and VSMC apoptosis in vitro. Knocking down BMAL1 decreased the level of anti-apoptotic protein BCL-2, while overexpressing BMAL1 could complement BCL-2 in VSMCs Figure 3 I-3K). Knocking down BMAL1 inhibited the cell viability of VSMCs Figure 3 L) and increased the level of TUNEL Figure 3 M-3N). Therefore, experiments at both mouse and cellular levels showed that BMAL1 deficiency activated VSMC apoptosis.

[0026] (4) BMAL1 activator ISX-9 reduces the incidence of TAAD Our previous work has confirmed that ISX-9 is an effective BMAL1 activator that can promote the transcription of BMAL1 mRNA and enhance its activity. Our data showed that ISX-9 increased the mRNA and protein levels of BMAL1 in mouse aortas Figure 4 A-4C). We administered ISX-9 to mice by intraperitoneal injection from the start of BAPN modeling and 14 days after modeling. Administering ISX-9 from the start of BAPN modeling significantly reduced the incidence of TAAD in BMAL1 knockdown mice Figure 5 A-5C). TranscriptomicsFigure 5 D-5G), immunofluorescence (F-5H), and Western blot (G-5I) all indicated that ISX-9 alleviated VSMC apoptosis in BMAL1 knockdown mice. Similarly, ISX-9 administration from the start of BAPN modeling significantly reduced the incidence of TAAD (K-5M) and alleviated VSMC apoptosis (L-5N) in wild-type mice. Notably, ISX-9 administration from 14 days after BAPN modeling also reduced the incidence of TAAD (P-5R) and alleviated VSMC apoptosis (Q-5S) in BMAL1 knockdown mice and wild-type mice. Importantly, ISX-9 administration did not significantly affect the body weight, blood pressure, and blood lipid levels of mice (O-5P), reflecting its safety and low side effects as a TAAD intervention drug. Figure 5 Figure 5 Figure 5 Figure 5 Figure 5 Figure 5 Figure 6

[0027] The above merely describes the preferred embodiments of the present application, and is not intended to limit the present application in any form or in essence. It should be noted that those skilled in the art can make several improvements and supplements without departing from the present application, and these improvements and supplements should also be considered as the protection scope of the present application.​​​​​​​

Claims

1. The use of BMAL1 activator ISX-9 in the preparation of drugs for the prevention and / or treatment of thoracic aortic aneurysms and dissections.

2. The application as described in claim 1, characterized in that, The drug comprises an active ingredient and pharmaceutically acceptable excipients or carriers, wherein the active ingredient comprises ISX-9.

3. The application as described in claim 1 or 2, characterized in that, The dosage forms of the drug include suspensions, tablets, granules, capsules, injections, powders, pills, suppositories, aerosols, or drops.

4. The application as described in claim 1 or 2, characterized in that, The routes of administration of the drug include intraperitoneal injection, intravenous injection, intramuscular injection, subcutaneous injection, oral administration, sublingual administration, nasal administration, or nebulized administration.

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