Application of succinate dehydrogenase complex B subunit as therapeutic target to preparation and screening of drugs for preventing or treating aortic valve calcification

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to application of a succinate dehydrogenase complex B subunit SDHB as a therapeutic target to preparation of drugs for screening and preventing and / or treating aortic valve calcification. The invention proves that the ROR alpha agonist has a protective effect on CAVD through an SDHB-mediated mechanism. In short, succinic acid in the calcified aortic valve is metabolized and accumulated, and in-vitro supplementation of succinic acid promotes osteogenesis and calcification of aortic valve interstitial cells (h-VICs); the overexpression of ROR alpha improves the abnormal mitochondrial metabolism of h-VICs; sDHB is intervened to counteract aortic valve calcification mediated by the ROR alpha agonist to abnormal mitochondrial metabolism. Therefore, as a potential therapeutic target for aortic valve calcification, SDHB can be used for screening and preparing drugs for treating or preventing CAVD, and the technical problem that treatment can only be carried out through a valve replacement operation at present, and an effective drug treatment means is lacked is solved.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and particularly to the use of succinate dehydrogenase complex subunit B as a therapeutic target in the preparation and screening of drugs for preventing or treating aortic valve calcification. Background Art

[0002] Calcific aortic valve disease (CAVD) is a heart valve disease mainly characterized by fibrosis, sclerosis, and calcium salt deposition in the aortic valve and its surrounding tissues, and is one of the common cardiovascular diseases. CAVD occurs through complex intracellular processes, including endothelial injury and dysfunction, immune cell infiltration, myofibroblast / osteoblast differentiation of aortic valve interstitial cells (VICs), and finally calcified matrix deposition. Although our understanding of this process has advanced, the mechanism by which VICs are transformed into osteoblasts remains unclear. Currently, the main treatment methods for aortic valve calcification are surgeries, such as aortic valve replacement, aortic decalcification, and percutaneous balloon valvuloplasty, etc., but they have disadvantages such as high risk, many complications, and poor late effects, and at the same time, they will also increase the economic burden on the patient's family. So far, there has been no safe and effective drug for preventing and inhibiting aortic valve calcification. Therefore, exploring drugs with high efficiency and few side effects for anti-aortic valve calcification has become an important direction in basic and clinical research.

[0003] The differentiation of VICs into myofibroblasts and osteoblasts is considered a key feature of the pathogenesis of CAVD. A large number of studies have shown that there is a strong correlation between the osteogenic differentiation of VICs and mechanical stress and metabolic changes. Research has shown that glycolysis plays an important role in the osteogenic differentiation of VICs. In addition, research results from different research groups have shown that the expression of several mitochondrial enzymes and metabolism-related genes is upregulated in calcified aortic valve tissues and VICs. Mitochondrial dysfunction is associated with the development of various heart diseases, such as atherosclerosis, hypertension, diabetes, and heart failure. Strict control of mitochondrial function is crucial for maintaining metabolic homeostasis. A recent metabolomics study provided strong evidence that inflammation and oxidative stress are the main molecular mediators regulating the degradation and remodeling process of CAVD. In addition, mitochondrial dysfunction and abnormal autophagy regulation are also associated with the injury of aortic valve interstitial cells (VICs). However, the application of mitochondrial metabolic disorders in aortic valve calcification has not been explored by anyone.

[0004] Succinate dehydrogenase (SDH) is an important enzyme complex located on the inner mitochondrial membrane and consists of SDHA, SDHB, SDHC, SDHD, and SDHAF2 subunits. It plays a key role in the mitochondrial respiratory chain, participates in the tricarboxylic acid (TCA) cycle, and catalyzes the conversion of succinate to fumarate. This reaction is a key step in aerobic oxidation and electron transfer. Any mutation in the subunits of the SDH complex may lead to functional impairment, resulting in the accumulation of succinic acid and reactive oxygen species (ROS). The accumulation of these compounds may competitively inhibit structurally similar α-ketoglutarate-dependent demethylases, thereby affecting gene expression regulation. In particular, the succinate dehydrogenase complex subunit B (SDHB), which is an important component of mitochondrial complex II, is crucial for catalyzing the conversion of succinate to fumarate. This conversion is essential for aerobic metabolism and the electron transport chain. SDHB also plays a key role in maintaining ion stability inside and outside cells, supporting important cellular activities within tissues. Mutations in the SDHB gene may disrupt the normal expression and function of tissues and cells, potentially affecting mitochondrial function and metabolic regulation. Given the central role of SDHB in these processes, its dysregulation may have a significant impact on cell health and overall metabolic homeostasis.

[0005] The inventors' research found that mitochondrial dysfunction is closely related to aortic valve calcification, and regulating mitochondrial function may be a potential therapeutic target. The present invention discovered a metabolite - succinic acid with potential for preventing and treating aortic valve calcification. Overexpression of the succinate dehydrogenase subunit SDHB can effectively inhibit osteogenic calcification of aortic valve interstitial cells and reduce valve calcification. SDHB overexpression can improve aortic valve calcification mediated by mitochondrial autophagy dysfunction. The succinate metabolism mediated by SDHB plays a role in alleviating aortic valve calcification by improving mitochondrial function. Based on this, the present invention provides an application of SDHB in the preparation of a drug for preventing or treating aortic valve calcification by regulating the metabolite succinic acid, so as to solve the technical problem that current clinical drugs for lipid-lowering and hemodynamic regulation cannot prevent the occurrence of aortic valve calcification or delay its progression. Summary of the Invention

[0006] Aiming at the technical problems of the prior art, the present invention provides an application of the succinate dehydrogenase complex subunit B as a therapeutic target in the preparation of a drug for screening for preventing or treating aortic valve calcification, so as to prevent and treat the occurrence of aortic valve calcification or delay its progression.

[0007] The present invention provides the following technical solutions:

[0008] Use of succinate dehydrogenase complex subunit B as a therapeutic target in the preparation and screening of drugs for preventing and / or treating aortic valve calcification in an individual.

[0009] Furthermore, the drug promotes the metabolism of succinic acid in the tricarboxylic acid cycle (TCA) by activating the overexpression of succinate dehydrogenase complex subunit B (SDHB), thereby inhibiting or slowing down the osteogenic calcification of aortic valve interstitial cells and reducing aortic valve calcification.

[0010] Furthermore, the inhibition or delay is achieved by activating the overexpression of succinate dehydrogenase complex subunit B (SDHB) to improve mitochondrial autophagy dysfunction-mediated aortic valve calcification.

[0011] Furthermore, the individual is a mammal.

[0012] Furthermore, the individual is a human.

[0013] The present invention also provides the use of an agonist of succinate dehydrogenase complex subunit B or a substance that improves succinic acid metabolism in the preparation of drugs for preventing and / or treating aortic valve calcification.

[0014] The present invention also provides the use of a combination of an agonist of succinate dehydrogenase complex subunit B or a substance that improves succinic acid metabolism in the preparation of drugs for preventing and / or treating aortic valve calcification.

[0015] Furthermore, the nuclear receptor RORα gene is located on the antisense strand of human chromosome 15, and its locus information is as follows: Homo sapiens chromosome 15, GRCh38.p14 Primary Assembly, NC_000015.10(60488284..61229302,complement); its transcript (NM_002943.4) (mRNA) sequence is as shown in SEQ ID NO.1. The agonist of the nuclear receptor RORα is SR1078 (chemical name: N-[4-[2,2,2-trifluoro-1-hydroxy-1-(trifluoromethyl)ethyl]phenyl]-4-(trifluoromethyl)benzamide), English name: N-[4-[2,2,2-Trifluoro-1-hydroxy-1-(trifluoromethyl)ethyl]phenyl]-4-(trifluoromethyl)benzamide; CAS number: 1246525-60-9; company: MedChemExpress,

[0016] The structural formula is:

[0017]

[0018] Furthermore, the drug prevents and / or treats aortic valve calcification by regulating the expression of succinate dehydrogenase complex subunit B and / or using nuclear receptor RORα.

[0019] The present invention also provides the use of a vector with overexpression of nuclear receptor RORα gene in the preparation of a drug for preventing and / or treating aortic valve calcification.

[0020] The present invention confirms that RORα agonist has a protective effect on CAVD through an SDHB-mediated mechanism. Briefly, succinate metabolism accumulates in calcified aortic valves, and in vitro supplementation of succinate promotes osteogenic calcification of aortic valve interstitial cells (h-VICs); overexpression of RORα improves the mitochondrial metabolic abnormalities of h-VICs; interfering with SDHB cancels the alleviating effect of RORα agonist on aortic valve calcification. As a key subunit of succinate dehydrogenase, SDHB regulates the downstream metabolites of succinate in the tricarboxylic acid cycle. Abnormality of SDHB leads to the accumulation of succinate, and the protective effect of overexpression of RORa on aortic valve calcification is cancelled by SDHB silencing. Therefore, SDHB, as a potential therapeutic target for aortic valve calcification, can be used to screen and prepare drugs for treating or preventing CAVD, solving the technical problem that currently only valve replacement surgery can be used for treatment and there is a lack of effective drug treatment means.

[0021] Compared with the existing technologies, the present invention provides new ideas and methods for the research on the pathogenesis, prevention and treatment of aortic valve calcification, and its technical effects are positive and obvious. On the one hand, the present invention finds that mitochondrial dysfunction is closely related to aortic valve calcification, and regulating mitochondrial function may be a potential therapeutic target, which is expected to become an important direction for future research. On the other hand, the present invention discovers a molecular target SDHB with potential for preventing and treating aortic valve calcification. The abnormal SDHB-mediated mitochondrial succinate metabolism disorder promotes osteogenic calcification of aortic valve interstitial cells, providing a new target for the pathophysiological mechanism of aortic valve calcification, solving the technical problem of non-surgical drug treatment of aortic valve calcification, providing an effective non-surgical drug treatment means for this disease, avoiding the risks and postoperative complications of valve replacement surgery, and having good application prospects. In addition, the present invention also explores the levels of mitochondrial intermediate metabolites in circulating cells as potential biomarkers for predicting pathological calcification, which is expected to become an important direction for future basic and clinical research. Brief Description of the Drawings

[0022] Figure 1 : Metabolomics analysis of CAVD and non-CAVD human aortic valve samples. A shows the differential metabolites in CAVD and non-CAVD group human aortic valve samples, B shows the metabolome analysis, and C shows the result graph of metabolite changes between CAVD and non-CAVD groups.

[0023] Figure 2 : Effects of succinic acid on osteogenic calcification of valvular interstitial cells. A and B are Western blot analysis and quantitative determination of the effects of succinic acid treatment on the expression of ALP, RUNX2, and BMP2 proteins. C and D are alizarin red staining for calcium deposition and calcium concentration determination to detect the effects of succinic acid on osteogenic calcification of valvular interstitial cells.

[0024] Figure 3 : Effects of SDHB silencing and overexpression of RORα on mitochondrial function and CAVD progression.

[0025] Figure 4 : Effects of SDHB silencing and RORα agonist SR1078 on mitochondrial function and CAVD progression. Specific implementation manners

[0026] The following further details the specific implementation manners and technical solutions of the present invention in conjunction with the accompanying drawings and specific embodiments. The specific embodiments give preferred implementation manners. In the present invention, unless otherwise specified, the raw materials can be purchased from conventional commercially available products, and the equipment and testing methods are conventional equipment and methods in the art.

[0027] Experimental animals: ApoE - / - , - / - , - / - mice were used. A calcific aortic valve disease (CAVD) model was induced by wire injury combined with 8-week high-fat diet (HFD). ApoE - / - mice of the same litter were subjected to sham operation as the control group. All experimental procedures for ApoE - / - mice complied with the guidelines stipulated in the "Guide for the Care and Use of Laboratory Animals" published by the National Institutes of Health (NIH publication, 8th edition, 2011). The study was approved by the Animal Ethics Committee of Shanghai Jiao Tong University, approval number: RJ2018-1018. Adult male mice aged 8 to 12 weeks were housed under standardized conditions. The breeding environment was strictly controlled at 24 ± 2 °C and humidity 40 ± 5% to ensure the health of the animals. The animals were housed in a pathogen-free environment with a 12-hour light / dark cycle. They had unrestricted access to food and water.

[0028] Molecular biochemical reagents: β-phosphoglycerate, dexamethasone, ascorbic acid, protease and phosphatase inhibitors, collagenase II type II, etc. were purchased from Sigma, USA; BCA quantitative kit was purchased from Thermo Scientific, USA; Tri-Buffered Saline (TBS), Tween-20, Phosphate-Buffered Saline (PBS), RORα primers and DAPI staining solution were purchased from Sangon Biotechnology (China); ALP, RUNX2, BMP2, TNF-α, IL-6, MCP-1, Caspase-3, Cleaved-Caspase-3, Bax, Bcl2 and other antibodies were purchased from abcam, USA; GAPDH antibody was purchased from Cell Signaling Technology, USA; chemiluminescent HRP substrate ECL luminescent solution was purchased from Millipore, USA; goat anti-mouse Ig G-HRP and goat anti-rabbit Ig G-HRP were purchased from Santa Clara, CA. cruz, USA, isoflurane was purchased from Runanbet, China, ELISA kits were purchased from R&Dsystem, USA, and RORα agonist SR1078 was purchased from MCE, USA.

[0029] Example 1: Establishment of mouse aortic valve calcification model.

[0030] Take 8-12 weeks old ApoE - / - In mice, aortic valve injury was induced using a wire transcarotid artery insertion technique. Specifically, the mice were anesthetized with isoflurane (flow rate 1.0 L / min, isoflurane concentration approximately 1.5%). Blunt dissection exposed the right carotid artery, and a spring wire (diameter 0.36 mm) bent at a 15-degree angle was inserted into the artery under the guidance of ultrasound echocardiography. The aortic valve was gently scratched 20 times with the spring wire, and rotated 100 times on the aortic valve to cause injury. Subsequently, the neck wound was rinsed with saline and then sutured. The right carotid artery was punctured through the left ventricle, and no spring wire was inserted as a control group. Aortic valve tissue was collected after 8 weeks of high-fat diet (HFD) after surgery.

[0031] Example 2: Establishment of an in vitro model of osteocalcification of aortic valve interstitial cells.

[0032] Human primary aortic valve interstitial cells (h-VICs) were isolated from the aortic valves of patients. Specifically, in aortic valve replacement surgery, the patient's aortic valve was first removed and immediately immersed in cold phosphate-buffered saline (PBS). Then, it was digested with 0.25% type II collagenase (Worthington, B1067, USA) at 37°C for 30 minutes. After initial digestion, the endothelium on the aortic and ventricular surfaces of the leaflets was gently scraped off with a cotton swab. Subsequently, the leaflets were cut into 1-2 mm slices and incubated in a 1 mg / ml collagenase solution at 37°C for 4-6 hours. Subsequently, the suspension was centrifuged at 1000 rpm for 10 minutes to form aggregates of h-VICs. Then, the tissue aggregates were broken by repeated aspiration. h-VICs were cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco) supplemented with 1% penicillin / streptomycin and 10% fetal bovine serum. The culture conditions were maintained in a humid environment at 37°C with 5% carbon dioxide. The cells were identified by immunofluorescence staining. h-VICs from passage 2 to passage 4 were induced using osteogenic medium (OM). The OM medium was prepared by adding 10 mM β-glycerophosphate, 10 nM dexamethasone, and 50 μg / mL ascorbic acid to DMEM respectively. After 14 days of OM induction, an in vitro model of osteogenic calcification of aortic valve interstitial cells was prepared.

[0033] Example 3: Metabolomics analysis in CAVD and non-CAVD human aortic valve samples.

[0034] To deepen the understanding of the metabolic profile during aortic valve calcification, targeted metabolomics analysis was performed on aortic valve tissues from patients diagnosed with CAVD and those not diagnosed with CAVD (Non-CAVD) using liquid chromatography-mass spectrometry. 142 metabolites were detected with different abundances between the two groups (P < 0.05, |log2 fold change| > 0.25). The detected metabolites were involved in central carbon metabolism, the citric acid cycle, and the cAMP signaling pathway. Notably, compared with non-CAVD patients, succinic acid in the aortic valve tissue of CAVD patients was significantly increased. Figure 1 A shows the differential metabolites in CAVD and non-CAVD groups of human aortic valve samples. Each column in Figure A represents an independent replicate experiment, and each row represents an independent metabolite. Upregulated metabolites are shown in red, and downregulated metabolites are shown in blue (n = 20 for both groups of samples). Figure 1Figure B shows that metabolome analysis indicates that the central carbon metabolic pathway is most significantly affected. Figure C is a volcano plot showing the significance and magnitude of metabolite changes between the CAVD and non-CAVD groups, using the same differential metabolite identification criteria as in Figure D. The differential metabolite succinic acid was identified through the above metabolomics analysis, revealing the important role of the central carbon metabolic pathway in CAVD.

[0035] Example 4: Effect of succinic acid on osteogenic calcification of valvular interstitial cells.

[0036] Since the phenotypic changes of h-VICs are closely related to mitochondrial metabolism, h-VICs were stimulated with the mitochondrial metabolite succinic acid, and Western blot analysis and quantitative determination showed that succinic acid treatment led to a significant increase in the expression of ALP, RUNX2, and BMP2 proteins ( Figure 2 Figures 2A and 2B). In addition, to study the direct role of succinic acid in the calcification process, we added succinic acid to the cell culture medium, and then detected calcium deposition by alizarin red staining and calcium concentration determination methods. This addition significantly increased the calcium content of h-VICs under OM stimulation ( Figure 2 Figures 2C and 2D). These results indicate that succinic acid may play a key role in the calcification of h-VICs.

[0037] Example 5: Effects of SDHB silencing and RORα overexpression on mitochondrial function and CAVD progression.

[0038] To investigate whether the protective mechanism of RORα overexpression (RORα-OE) on mitochondrial function is related to SDHB, h-VICs were transfected with viruses of EV or RORα-OE at a concentration of 1×10 6 genome units / ml (GUT / ml), and after gentle stirring and incubation for 3 hours, the virus-containing medium was replaced with fresh medium and further incubated for 24 hours according to the manufacturer's instructions; then, small interfering RNA (siSDHB) targeting SDHB mRNA ( Figure 3 Figures 3A and 3B). Silencing of SDHB led to a decrease in basal, maximal, and ATP-coupled oxygen consumption rates, exacerbated OM-induced mitochondrial DNA (mtDNA) damage, and counteracted the protective effect of RORα-OE on h-VICs ( Figure 3C-3G). In fact, dysfunctional mitochondria are recognized and selectively targeted for degradation through the process of mitophagy, which plays a crucial role in maintaining mitochondrial homeostasis. According to the standard protocol, the mitochondrial membrane potential (MMP) was evaluated using the JC-1 kit from Sigma-Aldrich and tetramethylrhodamine methyl ester perchlorate (TMRM), also from Sigma-Aldrich. After stimulation, the cells were incubated with JC-1 staining solution or TMRM at 37 °C for 20 minutes. After incubation, the cells were washed three times with the appropriate buffer, JC-1 (JC-1 staining buffer) or phosphate-buffered saline (PBS) (for TMRM). MMP analysis was performed using a fluorescence microscope produced by Olympus, Japan, and a flow cytometer produced by Beckman Coulter, USA. The fluorescence intensity indicating MMP was quantified using ImageJ software to evaluate mitochondrial function in the cells. The presence of mitophagy was confirmed by observing the co-localization of GFP-LC3 and Mito-Tracker Red. Our study found that RORα-OE counteracted the decrease in GFP-LC3-positive, Mito-Tracker red-positive cells ( Figure 3 H, 3I), restored the mitochondrial membrane potential (ΔΨm) ( Figure 3 J), and alleviated mitochondrial apoptosis.

[0039] Example 6: Effects of SDHB silencing and RORα agonist SR1078 on mitochondrial function and CAVD progression.

[0040] To determine whether our findings were a direct result of altered RORα or secondary to aortic valve calcification, we sought to study the effects of pharmacological manipulation of RORα in the current model. Previous results showed that overexpression of RORα alleviated the progression of CAVD. To investigate the role of SDHB in aortic valve calcification in vivo, we used adeno-associated virus subtype 2 containing shRNA (AAV2-sh-SDHB) or AAV2-scramble control (negative control vector, AAV2-scr) and injected them via the tail vein to silence the expression of SDHB in ApoE - / - mice, and then injected saline or SR1078 intraperitoneally twice a day for 8 weeks ( Figure 4 A), that is, the experimental groups were divided into: ApoE - / - +WI+Saline+AAV-scr; ApoE - / - +WI+Saline+AAV-SDHB; ApoE - / - +WI+SR1078+AAV-scr; ApoE - / -There were four groups: +WI, SR1078, AAV-SDHB. SDHB silencing exacerbated WI-induced aortic valve calcification and counteracted the decrease in osteogenic markers promoted by SR1078( Figure 4 B-4E). Related histological examinations showed that SDHB silencing eliminated the calcification attenuation and leaflet thickening caused by SR1078, which were confirmed by von Kossa staining and gross morphology analysis respectively( Figure 4 C-4F). In addition, in vivo multimodal imaging analysis including echocardiography, MRI and MUI enhanced the histopathological verification to confirm the effectiveness of MUI in evaluating aortic valve morphology and function( Figure 4 G). Inhibition of SDHB counteracted the improvement effects of SR1078 on transvalvular jet velocity, aortic orifice, aortic cusp separation and aortic valve area( Figure 4 H). Therefore, our results supported that overexpression of RORα had a protective effect on CAVD through SDHB-mediated mechanisms.

[0041] In summary, the present invention discovered a molecular target succinate dehydrogenase complex subunit B (SDHB) with potential for preventing and treating aortic valve calcification. Through a series of experiments, it was verified that succinate accumulation promoted osteogenic calcification of valvular interstitial cells, while activating succinate dehydrogenase subunit SDHB could effectively inhibit osteogenic calcification of aortic valvular interstitial cells, reduce valve calcification, and overexpression of SDHB could improve mitochondrial autophagy dysfunction-mediated aortic valve calcification, thereby inhibiting or delaying aortic valve calcification. The present invention further explored a new strategy for improving aortic valve calcification by regulating SDHB and using the RORα agonist SR1078, further revealing the pathogenesis of CAVD, and providing new targets, new methods and new ideas for developing novel therapeutic methods for CAVD.

[0042] The above are only specific embodiments of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art can easily think of changes or substitutions within the technical scope disclosed by the present invention, and all should be covered by the protection scope of the present invention. Therefore, the protection scope of the present invention should be subject to the protection scope of the claims.

Claims

1. Application of succinate dehydrogenase complex B subunit as a therapeutic target in the preparation and screening of drugs for preventing and / or treating aortic valve calcification.

2. The use according to claim 1, characterized in that: The drug stimulates the overexpression of succinate dehydrogenase complex B subunit SDHB, promotes the metabolism of succinate in the tricarboxylic acid cycle, thereby inhibiting or slowing down the osteocalcification of aortic valve interstitial cells and reducing aortic valve calcification.

3. The use according to claim 2, characterized in that: The inhibition or delay is to improve aortic valve calcification mediated by mitochondrial autophagy dysfunction by stimulating overexpression of succinate dehydrogenase complex B subunit SDHB.

4. Use of an agonist of the B subunit of the succinate dehydrogenase complex or a substance that promotes the improvement of succinate metabolism in the preparation of a drug for preventing and / or treating aortic valve calcification.

5. Use of a combination of an agonist of the B subunit of the succinate dehydrogenase complex or a substance that promotes improved succinate metabolism and a RORα agonist in the preparation of a drug for preventing and / or treating aortic valve calcification.

6. The use according to claim 5, characterized in that: The nuclear receptor RORα gene is located on the antisense strand of human chromosome 15, and its site information is Homo sapiens chromosome 15, GRCh38.p14 Primary Assembly, NC_000015.10 (60488284..61229302, complement), and its transcript NM_002943.4 sequence is shown in SEQ ID NO.

1.

7. The use according to claim 6, characterized in that: The agonist of the nuclear receptor RORα is SR1078, the chemical name is: N-[4-[2,2,2-trifluoro-1-hydroxy-1-(trifluoromethyl)ethyl]phenyl]-4-(trifluoromethyl)benzamide), CAS number: 1246525-60-9.

8. The use according to any one of claims 5-6, characterized in that: The drug prevents and / or treats aortic valve calcification by regulating the expression of succinate dehydrogenase complex B subunit and / or using the nuclear receptor RORα.

9. Use of a vector for overexpressing the nuclear receptor RORα gene in the preparation of a drug for preventing and / or treating aortic valve calcification.

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