Use of cthrc1 in preparation of drugs for hypertensive myocardial remodeling

Abstract

The application belongs to the technical field of biological medicine, and provides application of CTHRC1 in preparation of a drug for hypertensive myocardial remodeling. The application proves that CTHRC1 is significantly highly expressed in myocardial tissue and peripheral blood of a patient with hypertensive myocardial remodeling, and activates a non-canonical Wnt signaling pathway to up-regulate expression of RhoA, ROCK1, NFAT1 and TGF-β by interacting with a key ligand FZD2 of a myocardial Wnt receptor, thereby promoting hypertrophy of myocardial cells and activation of myocardial fibroblasts, and aggravating the process of myocardial remodeling, and solves the problem of lack of effective treatment targets for hypertensive myocardial remodeling in the prior art. The application provides that CTHRC1 can be used as a treatment target for hypertensive myocardial remodeling, and a drug developed by taking CTHRC1 as a target can effectively inhibit the process of myocardial remodeling, and is suitable for research and development and preparation of an anti-hypertensive myocardial remodeling drug.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of CTHRC1 in the preparation of drugs for myocardial remodeling in hypertension. Background Technology

[0002] Cardiac remodeling is the most common target organ damage in hypertension. Epidemiological surveys show that 10-30%, and even more than 50%, of patients with moderate to severe hypertension have varying degrees of cardiac structural and functional changes, with left ventricular remodeling being the most common. Cardiac remodeling refers to compensatory or decompensatory structural and functional changes in the heart under various physiological or pathological conditions. This includes macroscopic changes in heart size, systolic and diastolic function, as well as microscopic changes in myocardial gene expression levels, energy metabolism patterns, and the myocardial cell microenvironment. Studies show that left ventricular remodeling, as an early clinical symptom of many cardiovascular diseases, accompanies the entire pathological process of chronic heart failure and infarct arrhythmias.

[0003] Currently, although some non-coding RNAs and small molecule biomarkers related to myocardial remodeling have been discovered, [8] While myocardial remodeling involves cell signaling pathways, its pathogenesis remains unclear, and blood pressure control remains the primary treatment approach in clinical practice. Different types of antihypertensive drugs effectively reduce left ventricular weight, ventricular wall thickness, and inhibit centripetal remodeling in hypertensive patients by reducing pressure load and other pathways. However, epidemiological surveys show that even with long-term effective blood pressure control, some hypertensive patients still exhibit myocardial remodeling phenotypes, which is the biggest limitation of treating myocardial remodeling solely by controlling blood pressure.

[0004] Therefore, screening and identifying new factors that play an important regulatory role in the formation of myocardial remodeling and elucidating their mechanisms of action are of great significance for a deeper understanding of the pathogenesis of myocardial remodeling and for finding new intervention targets. Summary of the Invention

[0005] The purpose of this invention is to provide the application of CTHRC1 in the preparation of drugs for myocardial remodeling in hypertension, so as to solve the problem of lack of specific diagnostic markers and effective intervention targets for myocardial remodeling in the prior art.

[0006] To achieve the above objectives, the technical solution of the present invention is as follows: The present invention provides the application of CTHRC1 in the preparation of drugs for treating myocardial remodeling in hypertension.

[0007] Preferably, CTHRC1 promotes the formation of hypertensive myocardial remodeling by activating the non-classical Wnt signaling pathway.

[0008] Preferably, the activation of the non-classical Wnt signaling pathway is specifically manifested as an increase in the expression levels of RhoA, ROCK1, NFAT1, and TGF-β in myocardial tissue.

[0009] Preferably, the CTHRC1 overexpression enhances the expression of AngII-induced α-SMA in cardiomyocytes.

[0010] Preferably, the active ingredient of the drug is a CTHRC1 inhibitor.

[0011] Preferably, the drug further includes pharmaceutically acceptable excipients.

[0012] Preferably, the pharmaceutically acceptable excipient is selected from at least one of the following pharmaceutically acceptable solvents, solubilizers, cosolvents, emulsifiers, osmotic pressure regulators, stabilizers, suspending agents, coating materials, anti-adhesives, binding agents, penetration enhancers, pH adjusters, buffers, surfactants, absorbents, diluents, filter aids, and controlled-release materials.

[0013] Preferably, the drug includes various acceptable dosage forms.

[0014] The beneficial effects of this invention are:

[0015] 1. This study confirmed that CTHRC1 is a key precipitating factor for myocardial remodeling in hypertension.

[0016] 2. It is clear that the CTHRC1-nonclassical Wnt signaling pathway is an important regulatory axis for myocardial remodeling.

[0017] 3. It provides new therapeutic targets, offering a theoretical basis for the development of antihypertensive myocardial remodeling drugs. Attached Figure Description

[0018] Figure 1 This invention includes transcriptome sequencing (GSE133054) of human myocardial hypertrophy tissue and non-classical Wnt pathway analysis diagrams (A: Volcano plot of differentially expressed genes; B, C: Enrichment of non-classical Wnt pathway; D: Differential expression of CTHRC1; E: Correlation between CTHRC1 and pathway molecules).

[0019] Figure 2 In this invention, CTHRC1 expression was significantly increased in the myocardial tissue of patients with dilated cardiomyopathy; ** in the figure indicates P < 0.01.

[0020] Figure 3 This invention compares peripheral blood CTHRC1 levels in patients with hypertensive myocardial remodeling.

[0021] Figure 4This invention describes the cardiac function, morphology, pathology, and molecular detection of the mouse TAC myocardial remodeling model (A: cardiac function; B: cardiac morphology; C: heart-to-body ratio / heart-to-tibia ratio; D: WGA staining; E: Sirius red staining; F: ANP expression; * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001).

[0022] Figure 5 The expression of CTHRC1 and the non-classical Wnt pathway in a mouse myocardial remodeling model in this invention is shown in the figure (A: CTHRC1 expression; B: RhoA, ROCK1, NFAT1, and TGF-β expression; * indicates P < 0.05, ** indicates P < 0.01, and *** indicates P < 0.001).

[0023] Figure 6 This invention describes the effect of CTHRC1 overexpression on AngII-induced cardiomyocyte hypertrophy and fibroblast activation (A: AAV-9 overexpression efficiency; B: cardiomyocyte cytoskeleton staining; C: ANP expression; D: α-SMA expression; * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001).

[0024] Figure 7 This is the result of the interaction between CTHRC1 and FZD2 verified in this invention;

[0025] Figure 8 This is a schematic diagram illustrating the principle of how the interaction between CTHRC1 and FZD2 activates the non-classical Wnt signaling pathway to promote pathological myocardial remodeling in this invention. Detailed Implementation

[0026] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0027] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0028] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0029] Example 1: Population-level study of the relationship between peripheral blood CTHRC1 and myocardial remodeling in hypertension

[0030] 1. Clinical screening and methods

[0031] The diagnostic criteria for hypertension are: systolic blood pressure ≥140 mmHg and / or diastolic blood pressure ≥90 mmHg, whether under control or with medication.

[0032] The diagnostic criteria for myocardial hypertrophy are as follows: Echocardiography is used to examine the following myocardial structural and functional parameters: ① LVIDD / LVISD: Left ventricular end-diastolic diameter / left ventricular end-systolic diameter; ② IVST: Interventricular septal thickness; ③ PWTs: Left ventricular posterior wall thickness at end-systole; ④ LVM: Left ventricular mass. Substituting these parameters into the following formula:

[0033] LVM(g)=0.8×[1.04×(LVIDD+IVST+PWT)3-(LVID-(LVIDD) 3 +0.6LVM

[0034] The LVM value is obtained, and the ratio of the LVM value to BSA (body surface area) is defined as LVMBSA. For males: LVMBSA > 125 g / m² 2 And women with LVMBSA >120 g / m 2 This is defined as myocardial hypertrophy.

[0035] Exclusion criteria: During the case selection process, patients with concurrent tumors, severe metabolic diseases other than diabetes, valvular heart disease, congenital heart disease and ischemic cardiomyopathy, and hypertensive myocardial remodeling due to immune diseases were excluded.

[0036] Myocardial tissue transcriptome sequencing: Myocardial transcriptome data of person number GSE133054 were obtained from the GEO database, the fluorescence staining results were validated, and the CTHRC1-regulated signaling pathway was analyzed.

[0037] Immunofluorescence staining of myocardial tissue: Myocardial tissue was fixed with 4% paraformaldehyde, dehydrated, and then embedded in paraffin. 0.6 μM pathological sections were cut, dewaxed, and then stained with immunofluorescence. CTHRC1 antibody was labeled with a red fluorescent tag, and α-Actin was labeled with a green fluorescent tag. After staining, images were taken under a fluorescence microscope.

[0038] Targeted protein mass spectrometry detection: After sample preparation and protein digestion of peripheral blood serum samples, LC-MS / MS (DIA mode, Astral mass spectrometer) analysis was performed, and qualitative and quantitative analysis of peptides and proteins was conducted.

[0039] 2. Results Analysis

[0040] 2.1 CTHRC1 plays a regulatory role in myocardial remodeling.

[0041] To screen for protein factors that play an important regulatory role in myocardial remodeling, this invention analyzed transcriptome sequencing data (GSE133054) of myocardial tissue from patients with myocardial hypertrophy. Differentially expressed genes (|Log2 FoldChange| ≥ 1, q value < 0.05) were screened, and functional enrichment analysis was performed on these genes. The activation levels of the non-canonical Wnt signaling pathway were analyzed, and the expression correlation between CTHRC1 and molecules in the non-canonical Wnt signaling pathway was also analyzed.

[0042] The results showed that, compared with normal myocardial tissue (n=8), the expression levels of 2118 and 1279 genes, respectively, were significantly upregulated and downregulated in the myocardial tissue of patients with myocardial hypertrophy (n=8). Figure 1 (A). Functional enrichment of differentially expressed genes showed that the non-canonical Wnt signaling pathway was significantly activated in the myocardial tissue of patients with myocardial hypertrophy. Figure 1 China B and Figure 1 (C). Among the differentially expressed genes involved in the non-classical Wnt signaling pathway, CTHRC1 showed the highest statistical significance (Log2FoldChange = 1.63, qValue = 0.0019). Figure 1 Further research revealed that the expression levels of multiple signaling molecules in the non-canonical Wnt signaling pathway were significantly positively correlated with CTHRC1. Figure 1 (E). The above results indicate that CTHRC1 promotes the formation of hypertensive myocardial remodeling by regulating the non-classical Wnt signaling pathway.

[0043] To verify the expression of CTHRC1 in myocardial tissue, immunofluorescence staining was performed on myocardial tissue from patients with dilated cardiomyopathy and control patients. The results showed that the expression of CTHRC1 in the myocardial tissue of patients with dilated cardiomyopathy was significantly higher than that in the control group. Figure 2 ).

[0044] 2.2 Peripheral blood CTHRC1 levels were significantly increased in patients with myocardial remodeling compared to patients with simple hypertension.

[0045] This invention utilizes targeted protein mass spectrometry to detect peripheral blood CTHRC1 levels in 20 patients with hypertensive myocardial remodeling. Results showed that peripheral blood CTHRC1 levels were significantly higher in patients with myocardial remodeling compared to patients with simple hypertension. Figure 3 ).

[0046] Example 2: Animal-level study of the relationship between CTHRC1 and myocardial remodeling in hypertension

[0047] 1. TAC-based mouse model of myocardial remodeling

[0048] A mouse model of myocardial remodeling was established by aortic arch ligation (TAC). The specific steps were as follows: 12-week-old male C57BL / 6 mice were anesthetized and placed in a supine position. A 3-5 cm incision was made along the midline of the neck, and subcutaneous tissues such as the thyroid gland were dissected to expose the sternum. The sternum was cut approximately 3 mm to expose the aortic arch. A 27G needle was used as a suture, and the aorta was ligated using 6-0 sutures. After ligation, the suture was removed, and the sternum and skin were sutured. After the mice recovered, they were returned to their cages and housed in an SPF-grade animal facility. The mouse model was evaluated 4 weeks post-surgery.

[0049] Modeling criteria: a significant increase in left ventricular mass index (LVMI); echocardiography showing left ventricular wall thickening (increased LVPW, LVAW, and IVS), and decreased ejection fraction (EF) and fractional shortening (FS).

[0050] Four weeks after model establishment, cardiac ultrasound was used to evaluate the structure and function of the mouse heart, and the heart-body ratio (HW / BW) and heart-tibia ratio (HW / TL) were calculated. Myocardial tissue was fixed in 4% paraformaldehyde, dehydrated, embedded in paraffin, and sectioned. WGA staining and Sirius red staining were used to evaluate the degree of myocardial hypertrophy and fibrosis. Real-time PCR (7500, Thermo Fisher Scientific) was used to detect the expression level of the hypertrophy marker ANP in myocardial tissue. Western blotting was used to detect the expression levels of CTHRC1, RhoA, ROCK1, NFAT1 and TGF-β in myocardial tissue.

[0051] Western Blot analysis: Proteins were extracted from mouse myocardial tissue and mixed with loading buffer, then boiled at 100°C for 10 min. Electrophoresis was performed using a 10% SDS-PAGE gel at a constant voltage of 120 V until completion. Transfer was performed at a constant current of 300 mA for 80 min. After transfer, the membrane was blocked with 10% BSA at room temperature for 2 h, then primary antibody was added, and incubated overnight at 4°C. The membrane was washed three times with TBST, Hrp-labeled secondary antibody was added, and after incubation at room temperature for 2 h, images were taken using an automated imaging system.

[0052] The results showed that the cardiac contractile function of mice was significantly reduced ( Figure 4 In the middle A, significant changes occur in the heart structure ( Figure 4 In mice with myocardial remodeling, the heart-to-body ratio and heart-to-tibia ratio were significantly increased (in B). Figure 4 (C). At the pathological level, WGA staining and Sirius red staining were used to evaluate the level of myocardial hypertrophy and fibrosis in mice. The results showed that 4 weeks after TAC surgery, the degree of myocardial hypertrophy and fibrosis in mice was significantly increased compared with the control group. Figure 4 China E and Figure 4At the molecular level, the expression level of the myocardial hypertrophy marker ANP was significantly increased in the myocardial tissue of mice undergoing myocardial remodeling (F). Figure 4 (Middle F).

[0053] Furthermore, 4 weeks after TAC surgery, the expression level of CTHRC1 in the myocardial tissue of myocardial remodeling mice was significantly increased. Figure 5 (A). The expression levels of key signaling molecules RhoA, ROCK1, NFAT1, and TGF-β in the non-canonical Wnt signaling pathway were significantly increased in myocardial tissue of mice undergoing myocardial remodeling. Figure 5 (B) The above results indicate that the non-classical Wnt signaling pathway regulated by CTHRC1 is involved in the formation of myocardial remodeling.

[0054] Example 3: At the cellular level, overexpression of CTHRC1 exacerbates AngII-induced cardiomyocyte hypertrophy and cardiomyocyte fibroblast activation.

[0055] Primary cardiomyocytes and cardiomyocyte fibroblasts from neonatal suckling mice were isolated and cultured; CTHRC1 overexpression was achieved by transfecting cells with adeno-associated virus-9 (AAV-9). 48 h after transfection, 10... -6 A myocardial remodeling cell model was constructed by stimulating M-cells with angiotensin II (AngII). The degree of cardiomyocyte hypertrophy was evaluated by fluorescently labeled phalloidin staining, and the expression levels of cardiomyocyte hypertrophy marker ANP and cardiomyocyte fibroblast activation marker α-SMA were detected by Real-time PCR (7500, Thermo Fisher Scientific).

[0056] The results showed that, 48 h after AAV-9 infection of cardiomyocytes, the expression level of CTHRC1 in cardiomyocytes was significantly increased ( Figure 6 (A). In cardiomyocytes, 48 ​​h after AAV-9 infection, 10 -6 M's AngII induces cardiomyocyte hypertrophy, and the results showed that overexpression of CTHRC1 could aggravate AngII-induced cardiomyocyte hypertrophy. Figure 6 The expression of CTHRC1 (CTHRC1) and ANP in cardiomyocytes was compared, while overexpression of CTHRC1 alone had little effect on cell viability. Figure 6 (C). In cardiomyocytes, overexpression of CTHRC1 promotes AngII-induced fibroblast activation, mainly manifested in the further enhancement of AngII-induced α-SMA expression in cardiomyocytes by overexpression of CTHRC1. Figure 6 (D).

[0057] To clarify the mechanism by which CTHRC1 activates the non-canonical Wnt signaling pathway, this invention verified the interaction between CTHRC1 and the key Wnt receptor ligand FZD2 using Co-IP. Figure 7 The results showed that CTHRC1 can directly interact with FZD2, a key ligand of the Wnt receptor.

[0058] In summary, CTHRC1 is significantly overexpressed in myocardial tissue under pathological stimulation. It can specifically interact with the key Wnt pathway receptor FZD2, thereby activating the non-canonical Wnt signaling pathway, upregulating the expression of downstream key molecules such as RhoA, ROCK1, NFAT1, and TGF-β, and ultimately promoting pathological myocardial remodeling processes such as cardiomyocyte hypertrophy, myocardial fibroblast activation, and extracellular matrix deposition. This suggests that CTHRC1 is a key driver molecule regulating the development and progression of hypertensive myocardial remodeling. Figure 8 ).

[0059] The above-described embodiments are merely preferred embodiments of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the appended claims.

Claims

1. Application of CTHRC1 in the preparation of drugs for treating myocardial remodeling in hypertension.

2. The application according to claim 1, characterized in that, The CTHRC1 promotes the formation of hypertensive myocardial remodeling by activating the non-classical Wnt signaling pathway.

3. The application according to claim 1, characterized in that, The activation of the non-classical Wnt signaling pathway is specifically manifested as an increase in the expression levels of RhoA, ROCK1, NFAT1, and TGF-β in myocardial tissue.

4. The application according to claim 1, characterized in that, The CTHRC1 overexpression enhances AngII-induced α-SMA expression in cardiomyocytes.

5. The application according to claim 1, characterized in that, The active ingredient in the drug is a CTHRC1 inhibitor.

6. The application according to claim 1, characterized in that, The drug also includes pharmaceutically acceptable excipients.

7. The application according to claim 6, characterized in that, The pharmaceutically acceptable excipients are selected from at least one of the following pharmaceutically acceptable solvents, solubilizers, cosolvents, emulsifiers, osmotic pressure regulators, stabilizers, suspending agents, coating materials, anti-adhesives, binding agents, penetration enhancers, pH adjusters, buffers, surfactants, absorbents, diluents, filter aids, and sustained-release materials.

8. The application according to claim 1, characterized in that, The drug includes various acceptable dosage forms.

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