Use of succinate dehydrogenase complex b subunit as a therapeutic target in the preparation of a medicament for the prevention or treatment of aortic valve calcification

Abstract

The present application belongs to the technical field of biological medicine, and particularly relates to the use of succinate dehydrogenase complex B subunit (SDHB) as a therapeutic target in the preparation of a drug for screening, prevention and / or treatment of aortic valve calcification. The present application proves that RORα agonists have a protective effect on CAVD through the mechanism mediated by SDHB. In short, succinate metabolism accumulates in calcified aortic valves, and ex vivo supplementation of succinate promotes osteogenic calcification of aortic valve interstitial cells (h-VICs); RORα overexpression improves mitochondrial metabolic abnormalities of h-VICs; intervention of SDHB offsets the aortic valve calcification mediated by RORα agonists on mitochondrial metabolic abnormalities. Therefore, SDHB as a potential therapeutic target for aortic valve calcification can be used for screening and preparing a drug for treating or preventing CAVD, and solves the technical problem that current treatment can only be achieved through valve replacement surgery and there is a lack of effective drug treatment means.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to the use of the B subunit of succinate dehydrogenase complex as a therapeutic target in the preparation and screening of drugs for the prevention or treatment of aortic valve calcification. Background Technology

[0002] Calcific aortic valve disease (CAVD) is a common cardiovascular disease characterized by fibrosis, sclerosis, and calcium deposition in the aortic valve and surrounding tissues. CAVD develops through complex intracellular processes, including endothelial injury and dysfunction, immune cell infiltration, myofibroblast / osteoblast differentiation of aortic valvular interstitial cells (VICs), and calcification of the matrix. Although our understanding of this process has improved, the mechanism by which VICs are converted into osteoblasts remains unclear. Currently, the primary treatment for CAVD is surgery, such as aortic valve replacement, aortic decalcification, and percutaneous balloon valvuloplasty. However, these procedures have drawbacks, including high risks, numerous complications, and poor long-term outcomes, while also increasing the financial burden on patients and their families. To date, there are no safe and effective drugs for the prevention and inhibition of aortic valve calcification. Therefore, exploring highly effective drugs with minimal side effects to combat aortic valve calcification has become an important direction in basic and clinical research.

[0003] Differentiation of ventricular interstitial cells (VICs) into myofibroblasts and osteoblasts is considered a key feature of CAVD pathogenesis. Numerous studies have shown a strong correlation between osteogenic differentiation of VICs and mechanical stress and metabolic changes. Research indicates that glycolysis plays a crucial role in the osteogenic differentiation of VICs. Furthermore, findings from different research groups suggest that the expression of several mitochondrial enzymes and metabolism-related genes is upregulated in calcified aortic valve tissue 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 essential for maintaining metabolic homeostasis. A recent metabolomics study provides strong evidence that inflammation and oxidative stress are the main molecular mediators regulating CAVD degradation and remodeling processes. In addition, mitochondrial dysfunction and autophagy dysregulation are also associated with damage to aortic valve interstitial cells (VICs); however, the application of mitochondrial metabolic disorders in aortic valve calcification remains unexplored.

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

[0005] The inventors discovered that mitochondrial dysfunction is closely related to aortic valve calcification, and regulating mitochondrial function may be a potential therapeutic target. This invention identifies succinate, a metabolite with potential for preventing and treating aortic valve calcification. Overexpression of the succinate dehydrogenase subunit SDHB effectively inhibits osteogenic calcification of aortic valve interstitial cells, reducing valve calcification. SDHB overexpression can improve aortic valve calcification mediated by mitochondrial autophagy dysfunction. SDHB-mediated succinate metabolism exerts its alleviating effect on aortic valve calcification by improving mitochondrial function. Based on this, this invention provides the application of SDHB in the preparation of drugs for the prevention or treatment of aortic valve calcification by regulating its metabolite succinate, thereby addressing the technical problem that current clinical drug treatments such as lipid-lowering and hemodynamic regulation cannot prevent the occurrence of aortic valve calcification or delay its progression. Summary of the Invention

[0006] To address the technical problems of existing technologies, this invention provides the application of the succinate dehydrogenase complex B subunit as a therapeutic target in the preparation and screening of drugs for the prevention or treatment of aortic valve calcification, so as to prevent and treat the occurrence of aortic valve calcification or delay its progression.

[0007] This invention provides the following technical solutions:

[0008] Application of the B subunit of succinate dehydrogenase complex as a therapeutic target in the preparation and screening of drugs for the prevention and / or treatment of individual aortic valve calcification.

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

[0010] Furthermore, the inhibition or delay is achieved by improving aortic valve calcification mediated by mitochondrial autophagy dysfunction through overexpression of the succinate dehydrogenase complex B subunit SDHB.

[0011] Furthermore, the individual in question is a mammal.

[0012] Furthermore, the individual in question is a person.

[0013] The present invention also provides the use of agonists of the B subunit of succinate dehydrogenase complex or substances that promote improved succinate metabolism in the preparation of medicaments for the prevention and / or treatment of aortic valve calcification.

[0014] The present invention also provides the use of a combination of an agonist of the succinate dehydrogenase complex B subunit or a substance that promotes improved succinate metabolism in the preparation of a medicament for the prevention and / or treatment of aortic valve calcification.

[0015] Furthermore, the nuclear receptor RORα gene is located on the antisense strand of human chromosome 15, with the following site information: Homo sapiens chromosome 15, GRCh38.p14 Primary Assembly, NC_000015.10(60488284..61229302,complement); its transcript (NM_002943.4) (mRNA) sequence is 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); 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 the B subunit of the succinate dehydrogenase complex and / or by using the nuclear receptor RORα.

[0019] This invention also provides the use of vectors that overexpress the nuclear receptor RORα gene in the preparation of drugs for the prevention and / or treatment of aortic valve calcification.

[0020] This invention confirms that RORα agonists have a protective effect against CAVD through a mechanism mediated by SDHB. In short, succinate accumulates in calcified aortic valves, and in vitro succinate supplementation promotes osteogenic calcification of aortic valve interstitial cells (h-VICs); RORα overexpression improves mitochondrial metabolic abnormalities in h-VICs; and intervention with SDHB counteracts the alleviating effect of RORα agonists on aortic valve calcification. SDHB, as a key subunit of succinate dehydrogenase, regulates downstream metabolites of succinate in the tricarboxylic acid cycle. Abnormal SDHB leads to succinate accumulation, and the protective effect of RORα overexpression on aortic valve calcification is counteracted by SDHB silencing. Therefore, SDHB, as a potential therapeutic target for aortic valve calcification, can be used to screen and prepare drugs for the treatment or prevention of CAVD, solving the current technical challenge of only being able to treat CAVD through valve replacement surgery and lacking effective drug treatment methods.

[0021] Compared with existing technologies, this invention provides new ideas and methods for the study of the pathogenesis, prevention, and treatment of aortic valve calcification, with positive and significant technical effects. On the one hand, this invention has found a close relationship between mitochondrial dysfunction and aortic valve calcification, suggesting that regulating mitochondrial function may be a potential therapeutic target and a promising direction for future research. On the other hand, this invention has discovered SDHB, a molecular target with potential for the prevention and treatment of aortic valve calcification. Abnormal SDHB-mediated mitochondrial succinate metabolism disorders promote osteogenic calcification of aortic valve interstitial cells, providing a new target for the pathogenesis of aortic valve calcification. This solves the technical challenge of non-surgical drug treatment for aortic valve calcification, providing an effective non-surgical drug treatment for this disease, avoiding the risks and postoperative complications of valve replacement surgery, and showing promising application prospects. Furthermore, this 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 basic and clinical research. Attached Figure Description

[0022] Figure 1 Metabolomics analysis of aortic valve samples from CAVD and non-CAVD groups. A shows differentially expressed metabolites in aortic valve samples from CAVD and non-CAVD groups; B shows the metabolomics analysis; and C shows the metabolite changes between the CAVD and non-CAVD groups.

[0023] Figure 2 Effects of succinic acid on osteogenic calcification of valvular interstitial cells. A and B show the effects of succinic acid treatment on the expression of ALP, RUNX2, and BMP2 proteins, as determined by Western blot analysis and quantification. C and D show the effects of succinic acid on osteogenic calcification of valvular interstitial cells, as determined by Alizarin Red staining and calcium concentration assays.

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

[0025] Figure 4 Effects of SDHB silencing and the RORα agonist SR1078 on mitochondrial function and CAVD progression. Detailed Implementation

[0026] The specific embodiments and technical solutions of the present invention will be further described in detail below with reference to the accompanying drawings and specific examples. The specific examples provide preferred embodiments. In this invention, unless otherwise specified, all 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] Laboratory animals: ApoE strains bred by Shanghai Southern Model Organisms Center Co., Ltd. - / - A calcified aortic valve calcification (CAVD) model was established in mice by inducing filament injury combined with an 8-week high-fat diet (HFD). ApoE from littermates was used. - / - Mice underwent sham surgery as a control group. ApoE - / - All experimental procedures for mice were performed in accordance with the guidelines outlined in the *Laboratory Animal Care and Use Guidelines* (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 housing environment was strictly controlled at 24±2℃ and 40±5% humidity to ensure animal health. 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-glycerol phosphate, dexamethasone, ascorbic acid, protease and phosphatase inhibitors, collagenase II, etc. were purchased from Sigma-Aldrich (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 Biotech (China); ALP, RUNX2, BMP2, TNF-α, IL-6, MCP-1, Caspase-3, Cleaved-Caspase-3, Bax, Bcl2, etc. antibodies were purchased from Abcam (USA); GAPDH antibody was purchased from Cell Signaling Technology (USA); chemiluminescent HRP substrate ECL luminescence solution was purchased from Millipore (USA); goat anti-mouse Ig G-HRP and goat anti-rabbit Ig G-HRP were purchased from Santa Cruz Biotechnology (USA). Cruz (USA), isoflurane (Lunan Better China), ELISA kit (R&Dsystems, USA), and RORα agonist SR1078 (MCE, USA) were purchased from MCE.

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

[0030] ApoE was administered to infants aged 8-12 weeks. - / - In mice, aortic valve injury was induced using a transcarotid artery insertion technique with a wire. Specifically, mice were anesthetized with isoflurane (flow rate 1.0 L / min, isoflurane concentration approximately 1.5%). The right carotid artery was exposed by blunt dissection. Under echocardiographic guidance, a 15-degree angled spring wire (0.36 mm in diameter) was inserted into the artery, and the aortic valve was gently scratched 20 times with the spring wire and rotated 100 times on the aortic valve to induce injury. Subsequently, the neck wound was rinsed with saline and then sutured. Left ventricular puncture of the right carotid artery was performed without spring wire insertion as a control group. Aortic valve tissue was collected 8 weeks postoperatively after a high-fat diet (HFD).

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

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

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

[0034] To deepen our understanding of the metabolomic 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 varying 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, succinate levels were significantly increased in aortic valve tissues from CAVD patients compared to non-CAVD patients. Figure 1 Figure A shows the differentially expressed metabolites in human aortic valve samples from the CAVD and non-CAVD groups. 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). Figure 1Figure B shows that metabolomics analysis revealed the central carbon metabolic pathway was most significantly affected. Figure C, a volcano plot, illustrates the significance and magnitude of metabolite changes between the CAVD and non-CAVD groups, using the same differential metabolite identification criteria as Figure D. The differential metabolite succinate 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 phenotypic changes in h-VICs are closely related to mitochondrial metabolism, stimulation of h-VICs with the mitochondrial metabolite succinate, and Western blot analysis and quantification, showed that succinate treatment significantly increased the expression of ALP, RUNX2, and BMP2 proteins. Figure 2 (A and 2B). Furthermore, to investigate the direct role of succinic acid in the calcification process, we added succinic acid to the cell culture medium and then used Alizarin Red staining to measure calcium deposition and calcium concentration to detect that this addition significantly increased the calcium content of h-VICs under OM stimulation. Figure 2 (C and 2D). These results suggest 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) against mitochondrial function is related to SDHB, EV or RORα-OE virus was used at a concentration of 1×10⁻⁶. 6 h-VICs were transfected at a concentration of genomic units / Ml (GUT / Ml) and incubated with gentle agitation for 3 hours. The virus-containing medium was then replaced with fresh medium, and the cells were incubated for an additional 24 hours according to the manufacturer's instructions. Subsequently, small interfering RNA (siSDHB) was transfected with SDHB mRNA. Figure 3 SDHB silencing leads to decreased basal, maximal, and ATP-coupled oxygen consumption, exacerbates OM-induced mitochondrial DNA (mtDNA) damage, and counteracts the protective effect of RORα-OE on h-VICs. Figure 3(C-3G). In fact, dysfunctional mitochondria are identified and selectively targeted for degradation through mitophagy, which plays a crucial role in maintaining mitochondrial homeostasis. Mitochondrial membrane potential (MMP) was assessed using the Sigma-Aldrich JC-1 kit and tetramethylrhodamine perchlorate (TMRM), also from Sigma-Aldrich, according to standard protocols. After stimulation, cells were incubated at 37°C for 20 min with either JC-1 staining solution or TMRM. After incubation, cells were washed three times with appropriate buffers: JC-1 (JC-1 staining buffer) or phosphate-buffered saline (PBS) (TMRM). MMP analysis was performed using a fluorescence microscope (Olympus, Japan) and a flow cytometer (Beckman Coulter, USA). The fluorescence intensity indicating MMP was quantified using ImageJ software to assess mitochondrial function in 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 reduction in GFP-LC3-positive and Mito-Tracker red-positive cells. Figure 3 H, 3I), restored mitochondrial membrane potential (ΔΨm) ( Figure 3 J), and reduced 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 are a direct result of RORα alteration or secondary to aortic valve calcification, we sought to investigate the effects of pharmacological manipulation of RORα in the current model. Previous results have shown that RORα overexpression alleviates 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 an AAV2-scramble control (negative control vector, AAV2-scr) via tail vein injection in ApoE. - / - In mice, SDHB expression was silenced, followed by intraperitoneal injections of saline or SR1078 twice daily for 8 weeks. Figure 4 A), that is, the experimental component is: ApoE - / - +WI+Saline+AAV-scr;ApoE - / - +WI+Saline+AAV-SDHB;ApoE - / - +WI+SR1078+AAV-scr;ApoE - / -Four groups were identified: +WI, +SR1078, +AAV, and -SDHB. SDHB silencing exacerbated WI-induced aortic valve calcification and counteracted the SR1078-promoted reduction in osteogenic markers. Figure 4 B-4E). Related histological examinations showed that SDHB silencing eliminated SR1078-induced calcification attenuation and lobular thickening, confirmed by von Kossa staining and gross morphological analysis, respectively. Figure 4 C-4F). Furthermore, in vivo multimodal imaging analysis, including echocardiography, MRI, and MUI, enhanced histopathological validation to confirm the effectiveness of MUI in assessing aortic valve morphology and function. Figure 4 G). Inhibition of SDHB counteracted the improving effects of SR1078 on transvalvular jet velocity, aortic foramen, aortic apex dissociation, and aortic valve area. Figure 4 Therefore, our results support the protective effect of RORα overexpression against CAVD through a mechanism mediated by SDHB.

[0041] In summary, this invention has discovered a molecular target, the succinate dehydrogenase complex B subunit SDHB, which has the potential to prevent and treat aortic valve calcification (CAVD). A series of experiments verified that succinate accumulation promotes osteogenic calcification of valvular interstitial cells, while activating 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, thereby inhibiting or delaying aortic valve calcification. This invention further explores a novel strategy to improve aortic valve calcification by regulating SDHB and using the RORα agonist SR1078, further elucidating the pathogenesis of CAVD and providing new targets, methods, and ideas for developing novel treatments for CAVD.

[0042] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. Application of RORα agonist SR1078 in the preparation of drugs for the treatment of aortic valve calcification. The chemical name of RORα agonist SR1078 is: N-[4-[2,2,2-trifluoro-1-hydroxy-1-(trifluoromethyl)ethyl]phenyl]-4-(trifluoromethyl)benzamide), CAS number: 1246525-60-9.

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