Application of mitochondrial calcium transporter inhibitors in the preparation of drugs for treating vascular calcification

Abstract

This invention discloses the application of mitochondrial calcium single-transporter (MCU) inhibitors in the preparation of drugs for treating vascular calcification, belonging to the field of biomedical technology. Studies have found that in vitamin D3-induced acute vascular calcification models and chronic kidney disease (CKD)-related vascular calcification models, the expression level of MCU in vascular tissue is significantly upregulated. The MCU inhibitor Ru360 can significantly reduce vascular calcium ion deposition, decrease Alizarin Red and Von Kossa staining positive areas, inhibit osteoblast-like phenotype transformation of vascular smooth muscle cells, and downregulate the expression of osteogenic transdifferentiation markers BGLAP and RUNX2. This invention provides a novel therapeutic target and candidate drug for vascular calcification, with broad clinical application prospects.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to a new pharmaceutical use of mitochondrial calcium single-transporter (MCU) inhibitors, particularly in the preparation of drugs for treating vascular calcification. Background Technology

[0002] Vascular calcification is an active and modifiable pathological process characterized by the pathological deposition of calcium phosphate crystals in the blood vessel walls. Vascular calcification is not only a common complication of atherosclerosis, hypertension, diabetes, and aging, but also an independent risk factor for increased cardiovascular event incidence and mortality in patients with chronic kidney disease (CKD). Epidemiological data show that as CKD progresses, decreased vascular compliance due to calcium and phosphorus metabolism disorders significantly increases the residual risk of cardiovascular disease.

[0003] Currently, clinical intervention strategies for vascular calcification mainly revolve around controlling hyperphosphatemia, regulating lipid metabolism, and using phosphate binders (such as sevelamer). However, to date, no drug specifically targeting the pathological mechanism of vascular calcification has been approved for marketing by the FDA or NMPA globally. The limitations of existing therapies are: while statins (such as atorvastatin) can stabilize plaques, they cannot reverse existing calcification; and conventional calcium channel blockers have insufficient tissue selectivity in regulating calcium metabolism. Therefore, in-depth exploration of the molecular mechanisms of vascular calcification and the development of therapeutic drugs with novel targets are urgent technical challenges that need to be addressed in the cardiovascular field.

[0004] Recent studies have confirmed that osteoblast-like phenotype transformation in vascular smooth muscle cells (VSMCs) is the cellular basis of vascular calcification. Mitochondrial dysfunction plays a crucial role as an upstream initiator in this process. Current techniques have revealed that a high-phosphorus environment-induced burst of mitochondrial reactive oxygen species (mtROS) and energy metabolism reprogramming are the core drivers of VSMC transdifferentiation. In particular, the imbalance of mitochondrial calcium homeostasis is considered a bridge connecting metabolic disorders and apoptosis. The mitochondrial calcium uniquer (MCU) is a transmembrane protein complex located on the inner mitochondrial membrane. It is the main channel for cytoplasmic calcium ions to enter the mitochondrial matrix and plays a decisive role in maintaining ATP synthesis, regulating the apoptosis threshold, and controlling oxidative stress responses.

[0005] Although literature reports that aberrant expression of micromolecular oxidase (MCU) is closely related to cardiovascular diseases such as myocardial ischemia-reperfusion injury, arrhythmia, and heart failure, the specific mechanism of MCU-mediated mitochondrial calcium homeostasis in vascular calcification (especially CKD-related vascular calcification) remains unclear. Currently, there is no evidence that MCU can serve as a therapeutic target for vascular calcification, nor are there any reports of MCU-specific inhibitors being used to treat vascular calcification. Existing known MCU inhibitors, such as ruthenium red and its derivative ruthenium 360 (Ru360), have shown potential in neuroprotection and arrhythmia studies, but their applicability as anti-vascular calcification drugs remains to be confirmed due to issues such as low bioavailability, high cytotoxicity, and low target specificity.

[0006] To address the shortcomings of the existing technologies, this invention reveals for the first time that MCU expression levels are significantly upregulated in CKD and VD3-induced calcified vascular tissues. Based on the pathological axis of "mitochondrial calcium disorder-energy impairment-osteogenic differentiation," it provides a novel pharmaceutical application for MCU inhibitors, thereby offering a new strategy and direct pharmacological evidence for the treatment of CKD-related vascular calcification. Summary of the Invention

[0007] The purpose of this invention is to provide a new pharmaceutical use for mitochondrial calcium single-transporter (MCU) inhibitors to address the lack of therapeutic drugs in the prior art that specifically target the pathological mechanism of vascular calcification.

[0008] To achieve the above objectives, this invention employs a vitamin D3 (VD3)-induced acute vascular calcification model in mice and a CKD-related vascular calcification model induced by 5 / 6 nephrectomy combined with a high-phosphorus diet. The degree of vascular calcification is assessed by measuring calcium ion content, alizarin red staining, and Von Kossa staining. The expression of MCU and osteogenic transdifferentiation markers (Runx2, BGLAP, RUNX2) is detected by qRT-PCR, Western blot, and immunohistochemistry. The direct effect of Ru360 is verified by in vitro aortic ring culture.

[0009] The results showed that in the CKD-related vascular calcification model, the mRNA and protein expression levels of MCU were significantly upregulated. In the VD3-induced vascular calcification model, intraperitoneal injection of Ru360 significantly reduced calcium ion content in the abdominal aorta, decreased the positive areas of Alizarin Red and Von Kossa staining, and downregulated the protein expression of the osteogenic metaplasia marker BGLAP. In the CKD-related vascular calcification model, Ru360 also significantly reduced calcium ion deposition, reduced calcification area, inhibited the mRNA expression of Runx2, Sox9, and Bglap, upregulated α-Sma expression, and decreased the protein levels of RUNX2 and BGLAP. In vitro aortic ring culture confirmed that Ru360 can directly act on vascular tissue to inhibit high-phosphorus-induced calcification.

[0010] Therefore, MCU is upregulated in CKD-related vascular calcification, making it a novel target for the treatment of vascular calcification. MCU-specific inhibitor Ru360 effectively reduces vascular calcification by inhibiting osteoblast-like phenotype transformation of vascular smooth muscle cells, thus providing a new drug candidate for the treatment of vascular calcification.

[0011] In a first aspect, the present invention provides the use of mitochondrial calcium single-transporter (MCU) inhibitors in the preparation of drugs for treating vascular calcification.

[0012] As a preferred embodiment, the mitochondrial calcium transtransporter inhibitor is ruthenium 360 (Ru360). However, the medicament of the present invention is not limited to Ru360, and other MCU inhibitors known in the art are also available, such as ruthenium red, DS16570511, mitoxantrone, KB-R7943, CGP 37157, berberine, etc.

[0013] As a preferred embodiment, the vascular calcification includes vascular calcification associated with chronic kidney disease (CKD), as well as acute vascular calcification caused by other reasons.

[0014] As a preferred embodiment, the drug comprises a therapeutically effective amount of a mitochondrial calcium oneway transporter inhibitor and a pharmaceutically acceptable carrier.

[0015] Furthermore, the dosage form of the drug is an injection.

[0016] The beneficial effects of this invention are: This invention confirms that micromolecular calcification (MCU) is a novel and effective target for treating vascular calcification. It is the first time that the known MCU-specific inhibitor Ru360 has been used to prepare an anti-vascular calcification drug, expanding the clinical application scope of Ru360 and providing a therapeutic strategy targeting mitochondrial calcium homeostasis for vascular calcification. This invention also elucidates the molecular mechanism by which Ru360 treats vascular calcification by restoring mitochondrial homeostasis and blocking the "calcium disorder-osteogenic differentiation" axis. It confirms that Ru360 acts directly on vascular tissue, rather than through systemic metabolic regulation, providing direct evidence for its potential as a local or systemic therapeutic agent targeting blood vessels, and demonstrating high clinical translational potential. Attached Figure Description

[0017] Figure 1 The graph shows the results of calcium ion content detection in the abdominal aorta of mice in each group in Example 1.

[0018] Figure 2 The image shows the results of alizarin red staining of the gross arterial tissues of mice in each group in Example 1.

[0019] Figure 3 The image shows the Von Kossa staining results of aortic arch tissue sections from each group of mice in Example 1, with a scale bar of 50 μm.

[0020] Figure 4 The image shows the Western blot results of BGLAP protein detection in the vascular tissues of mice in Example 1.

[0021] Figure 5 The image shows the qRT-PCR detection results of the Mcu gene in the vascular tissue of the CKD model mouse in Example 2.

[0022] Figure 6 This is a Western blot result of MCU protein detection in vascular tissue of CKD model mice in Example 2.

[0023] Figure 7 The image shows the immunohistochemical staining and quantitative analysis results of MCU in the vascular tissue of the CKD model mouse in Example 2. Scale bar = 50 μm.

[0024] Figure 8 The image shows the results of calcium ion content detection in the abdominal aorta of CKD model mice in Example 2.

[0025] Figure 9 This is a diagram showing the results of alizarin red staining of gross arterial tissue from CKD model mice in Example 2.

[0026] Figure 10 The image shows the Von Kossa staining results of aortic arch tissue sections from CKD model mice in Example 2. Scale bar = 50 μm.

[0027] Figure 11 The image shows the qRT-PCR detection results of osteogenic differentiation marker genes and contraction phenotype marker genes in the vascular tissue of CKD model mice in Example 2.

[0028] Figure 12 The image shows the results of Western blot analysis of RUNX2 and BGLAP proteins in the vascular tissue of CKD model mice in Example 2.

[0029] Figure 13 The image shows the results of Von Kossa staining in the in vitro aortic ring culture in Example 2, with a scale bar of 50 μm.

[0030] In the above figures, the markings *P<0.05, **P<0.01, ***P<0.001, and ****P<0.0001 are used. Detailed Implementation

[0031] The present invention will now be described in detail with reference to specific embodiments. It should be noted that the specific embodiments described are merely illustrative of the invention, and the scope of protection of the invention is not limited to the following embodiments. Any changes and modifications made to the invention by those skilled in the art without departing from the concept of the invention should fall within the scope of protection of the invention. Furthermore, various experimental operations not described in detail in the embodiments are conventional techniques in the art. For parts not specifically described herein, those skilled in the art can refer to various commonly used reference books, scientific and technological documents, or related instructions and manuals prior to the filing date of this invention for implementation.

[0032] Key materials: SPF-grade 7-week-old male C57BL / 6 mice (approximately 18-20g) were purchased from Beijing Huafukang Biotechnology Co., Ltd., and housed in an SPF-grade barrier environment at the Tongji Medical College Animal Facility of Huazhong University of Science and Technology. Sterile feed and drinking water for the mice were provided by this animal facility. Ru360 was purchased from Sigma, catalog number 557440.

[0033] Example 1: Therapeutic effect of Ru360 on VD3-induced vascular calcification 1. Test Methods 1.1 Laboratory Animals and Grouping Seven-week-old male SPF-grade C57BL / 6 mice (approximately 18-20g) were selected and housed in an SPF-grade barrier environment. Sterile feed and drinking water were provided by the animal facility. The mice were randomly divided into four groups: control + PBS group (Ctrl+PBS group); control + Ru360 group (Ctrl+Ru360 group); VD3 + PBS group; and VD3 + Ru360 group, with six mice in each group.

[0034] 1.2 Establishment of a VD3-induced vascular calcification model The preparation method of VD3 solution is as follows: Weigh 33 mg of VD3 powder into a 50 mL centrifuge tube, add 200 µL of anhydrous ethanol, then add 1.4 mL of polyoxyethylene castor oil and mix thoroughly. Let stand for 15 min. Separately, place 750 mg of glucose into another 50 mL centrifuge tube, add 18.4 mL of PBS solution to dissolve the glucose, let stand for 15 min, and then mix the two solutions. Subcutaneously inject the VD3 solution into mice for 3 consecutive days, with an injection dose of 4 × 10⁻⁶ mg / mL. 5 IU / kg. Mice were sacrificed on day 7 after injection for tissue collection.

[0035] 1.3 Ru360 Dosing Regimen Ru360 was purchased from Sigma (catalog number 557440). Preparation method: 500 µg of Ru360 dry powder was added to 1 mL of autoclaved ddH2O to obtain a stock solution with a concentration of 907.8 µM. The solution was then aliquoted and stored frozen at -30°C. Light should be avoided during preparation. Intraperitoneal injections of Ru360 were initiated one week before the start of subcutaneous VD3 injections at a dose of 240 µg / kg, administered every two days until the mice were sacrificed.

[0036] 1.4 Detection Indicators (1) Determination of abdominal aortic calcium content: Take some abdominal aortic tissue from each group of mice, add 0.6 mM dilute hydrochloric acid, grind with a tissue grinder, extract on a shaker at 4℃ for 24 h, centrifuge and take the supernatant, use a calcium ion detection kit to measure the OD value at a wavelength of 610 nm, and at the same time use the BCA method to determine the protein concentration and calculate the standardized calcium ion deposition amount.

[0037] (2) Alizarin Red staining of gross tissue: The intact gross tissue of blood vessels was fixed in paraformaldehyde for 24 h, and then placed in 0.004% alizarin red staining solution overnight (16 h). After washing three times with 2% KOH solution, the tissue was photographed and recorded.

[0038] (3) Von Kossa staining of vascular tissue: take paraffin sections of aortic arch tissue, dewax and rehydrate them, add Von Kossa silver staining solution, stain under ultraviolet light for 1-2 h, stop staining with hyaluronic acid solution, stain the nuclei with hematoxylin, and observe after mounting.

[0039] (4) Western blot detection: Proteins were extracted from mouse vascular tissue, and after SDS-PAGE electrophoresis, membrane transfer and blocking, BGLAP primary antibody (dilution ratio 1:400) was added and incubated overnight at 4℃. Secondary antibody was incubated at room temperature for 1 h. ECL exposure imaging was performed, and grayscale analysis was performed using ImageJ software.

[0040] 2. Test Results 2.1 Ru360 reduces VD3-induced calcium ion content in the abdominal aorta of mice. The calcium ion content in the abdominal aorta of mice in each group was measured, and the results are as follows: Figure 1 As shown, compared with the control + PBS group, the amount of calcium ion deposition in the abdominal aorta of mice in the VD3 + PBS group was significantly increased (P<0.0001), indicating that the VD3-induced vascular calcification model in mice was successfully established; while the calcium ion content in the abdominal aorta of mice treated with Ru360 was significantly lower than that in the VD3 + PBS group (P<0.001).

[0041] 2.2 Ru360 reduces the area of ​​positive Alizarin Red staining in the gross angiography of mice induced by VD3. Alizarin red staining was performed on the gross arterial tissues of each group of mice, and the results are as follows: Figure 2 As shown: The blood vessels of mice in the control + PBS group and the control + Ru360 group were clear and transparent, with no obvious staining; the blood vessels of mice in the VD3 + PBS group showed a large number of purplish-red staining positive areas, indicating a large amount of calcium salt deposition; while the degree of calcification of blood vessels of mice in the VD3 + Ru360 group was significantly reduced, and the area of ​​alizarin red staining positive area was significantly reduced.

[0042] 2.3 Ru360 alleviates VD3-induced calcification of the aortic arch in mice Von Kossa staining was performed on sections of aortic arch tissue, and the results are as follows: Figure 3 As shown: no obvious black staining was observed in the blood vessels of mice in the control + PBS group and the control + Ru360 group; obvious black calcium salt deposits were visible in the blood vessels of mice in the VD3 + PBS group, indicating vascular calcification; while the degree of vascular calcification was significantly reduced in the VD3 + Ru360 group, and the black staining area was significantly reduced. This result is consistent with the results of alizarin red staining of gross vascular tissue.

[0043] 2.4 Ru360 downregulates VD3-induced BGLAP protein expression Proteins were extracted from vascular tissues of mice in each group and analyzed by Western blot. The results are as follows: Figure 4 As shown: Compared with the control + PBS group, the protein expression level of the osteogenic transdifferentiation marker BGLAP in the blood vessels of mice in the VD3 + PBS group was significantly upregulated (P<0.05), indicating that VD3 induces osteogenic transdifferentiation of VSMCs; while the BGLAP protein expression level in mice in the VD3 + Ru360 group injected intraperitoneally was significantly lower than that in the VD3 + PBS group (P<0.05).

[0044] Conclusion: The above results indicate that in a VD3-induced acute vascular calcification model in mice, intraperitoneal injection of Ru360 can effectively reduce vascular calcium ion content, decrease calcium salt deposition, alleviate the severity of vascular calcification, and inhibit osteogenic transdifferentiation of VSMCs, suggesting that Ru360 has a therapeutic effect on VD3-induced vascular calcification.

[0045] Example 2: Therapeutic effect of Ru360 on CKD-related vascular calcification 1. Test Methods 1.1 Establishment of a CKD-related vascular calcification model A CKD-related vascular calcification model was induced using a combination of 5 / 6 nephrectomy and a high-phosphorus diet, with specific methods described in reference (CN 120154629A). The simplified steps are as follows: Seven-week-old male C57BL / 6 mice were anesthetized intraperitoneally with 1% sodium pentobarbital. In the first week, a total left nephrectomy was performed, followed by a 2 / 3 right nephrectomy in the second week. After one week of postoperative recovery, a gradient high-phosphorus diet (1% high-phosphorus diet for 4 weeks, followed by a 2% high-phosphorus diet for 8 weeks) was administered to induce vascular calcification.

[0046] 1.2 Laboratory Animals and Grouping The model mice were randomly divided into the following four groups (n=6 per group): Sham+PBS group (sham surgery, intraperitoneal injection of PBS); Sham+Ru360 group (sham surgery, intraperitoneal injection of Ru360); 5 / 6Nx+PBS group (model control, intraperitoneal injection of PBS); 5 / 6Nx+Ru360 group (model, intraperitoneal injection of Ru360).

[0047] 1.3 Ru360 Dosing Regimen Ru360 was prepared as in the previous example. Intraperitoneal administration was initiated simultaneously with the start of a 1% high-phosphorus diet at a dose of 240 µg / kg, administered every 2 days for a total of 8 weeks. The control group received an equal volume of PBS.

[0048] 1.4 MCU Expression Detection qRT-PCR: Total RNA was extracted from the thoracic aorta of mice in each group, and the expression level of Mcu gene mRNA was detected after reverse transcription, with Gapdh as an internal control.

[0049] Western blot: Proteins were extracted from vascular tissue, subjected to SDS-PAGE electrophoresis, transferred to a membrane, incubated overnight at 4°C with MCU primary antibody (1:400), incubated at room temperature for 1 h with secondary antibody, ECL imaging, and the gray value was quantified using ImageJ software.

[0050] Immunohistochemical staining: After dewaxing and antigen retrieval of paraffin sections of vascular tissue, add MCU primary antibody (1:400) and incubate overnight at 4℃. Incubate with secondary antibody at room temperature for 30 min, develop DAB staining, counterstain with hematoxylin, and observe and quantify under a microscope.

[0051] 1.5 Detection of the therapeutic effect of Ru360 (1) Assessment of the degree of vascular calcification: Calcium ion content determination: Abdominal aortic tissue was extracted with dilute hydrochloric acid and the OD value was measured at a wavelength of 610 nm using a calcium ion detection kit. Protein concentration was determined by the BCA method, and the standardized calcium ion deposition was calculated.

[0052] Gross Alizarin Red staining: After the intact aorta is fixed, it is stained overnight in 0.004% Alizarin Red staining solution, washed, and photographed to observe the degree of calcification.

[0053] Von Kossa staining: After dewaxing the paraffin sections of the aortic arch, Von Kossa silver staining solution was added, and the sections were stained under ultraviolet light for 1-2 hours. Hematoxylin was then used for counterstaining, and calcium salt deposition was observed under a microscope.

[0054] (2) VSMC osteogenic transdifferentiation detection: qRT-PCR: Detect the mRNA expression levels of osteogenic differentiation marker genes Runx2, Sox9, Bglap, and VSMC contraction phenotype marker gene α-Sma in vascular tissue.

[0055] Western blot: Detect the expression levels of RUNX2 and BGLAP proteins.

[0056] (3) In vitro aortic ring culture: Aortic rings of C57BL / 6 mice were extracted and subjected to high phosphorus stimulation in vitro while Ru360 was added for intervention. The culture medium was changed every other day. After 2 weeks, the rings were embedded in paraffin and sectioned, and Von Kossa staining was performed to observe the direct effect of Ru360 on vascular tissue.

[0057] 2. Test Results 2.1 Upregulation of MCU expression in CKD-related vascular calcification To clarify the expression changes of MCU in CKD-related vascular calcification, vascular tissues of mice in the model group and sham-operated group were examined.

[0058] qRT-PCR results ( Figure 5 The results showed that, compared with the Sham group, the mRNA expression level of the Mcu gene in the vascular tissue of mice in the 5 / 6Nx+PBS group was significantly upregulated (P<0.01).

[0059] Western blot results ( Figure 6 The results showed that, compared with the Sham group, the expression level of MCU protein in the vascular tissue of mice in the 5 / 6Nx+PBS group was significantly increased (P<0.01).

[0060] Immunohistochemical staining results ( Figure 7The results showed that MCU staining was weak in the vascular tissue of mice in the Sham group, while obvious MCU-positive brown staining was visible in the vascular media and intima of mice in the 5 / 6Nx+PBS group. Quantitative analysis showed that the difference between the two groups was statistically significant (P<0.01).

[0061] The above results indicate that MCU expression levels are significantly upregulated in CKD-related vascular calcification.

[0062] 2.2 Ru360 reduces vascular calcification in CKD mice To verify the therapeutic effect of the MCU inhibitor Ru360 on vascular calcification, model mice were treated with intraperitoneal injection of Ru360.

[0063] Calcium ion content determination results ( Figure 8 The results showed that compared with the Sham+PBS group, the calcium ion deposition in the abdominal aorta of mice in the 5 / 6Nx+PBS group was significantly increased (P<0.001); while the calcium ion content in mice in the 5 / 6Nx+Ru360 group was significantly lower than that in the 5 / 6Nx+PBS group (P<0.01), indicating that Ru360 can effectively reduce intravascular calcium salt deposition.

[0064] Gross alizarin red staining results ( Figure 9 The results showed that the blood vessels in the Sham group were transparent; the blood vessels in the 5 / 6Nx+PBS group showed a large area of ​​uniformly distributed purplish-red staining, indicating extensive calcium salt deposition; while the purplish-red staining area in the blood vessels of the 5 / 6Nx+Ru360 group was significantly reduced, and the degree of calcification was significantly reduced.

[0065] Von Kossa staining results ( Figure 10 The results showed that no black calcium salt particles were observed in the blood vessels of the Sham group; a large number of black granular calcium salt deposits were observed in the vascular media of the 5 / 6Nx+PBS group; and the black staining area was significantly reduced in the 5 / 6Nx+Ru360 group, indicating a significant reduction in the severity of vascular calcification.

[0066] 2.3 Ru360 inhibits osteogenic transdifferentiation of VSMC To further clarify the molecular mechanism by which Ru360 alleviates vascular calcification, the expression changes of osteogenic transdifferentiation markers in VSMCs were detected.

[0067] qRT-PCR results ( Figure 11The results showed that, compared with the Sham group, the mRNA expression levels of osteogenic differentiation marker genes Runx2, Sox9, and Bglap in the vascular tissue of mice in the 5 / 6Nx+PBS group were significantly increased (P<0.01), and the mRNA expression level of ALPL (alkaline phosphatase) was also significantly increased (P<0.01), while the mRNA expression level of α-Sma, a VSMC contraction phenotype marker gene, was significantly decreased (P<0.01). These results indicate that osteogenic phenotypic conversion of VSMCs occurred in CKD-related vascular calcification.

[0068] Western blot results ( Figure 12 The results showed that, compared with the Sham+PBS group, the expression levels of RUNX2 and BGLAP proteins in the blood vessels of mice in the 5 / 6Nx+PBS group were significantly upregulated (P<0.001); while the content of RUNX2 and BGLAP proteins in the blood vessels of mice in the 5 / 6Nx+Ru360 group was significantly reduced compared with the 5 / 6Nx+PBS group (P<0.05, P<0.001).

[0069] The above results suggest that Ru360 reduces CKD-related vascular calcification by inhibiting osteogenic transdifferentiation of VSMCs.

[0070] 2.4 Ru360 directly inhibits calcification in vascular tissue. To verify whether Ru360 acts directly on vascular tissue rather than through systemic metabolic regulation, an in vitro aortic ring culture experiment was conducted.

[0071] result( Figure 13 The results showed that under in vitro high-phosphorus stimulation, significant calcium salt deposition (positive Von Kossa staining) occurred in the aortic rings; however, the addition of Ru360 significantly reduced the degree of calcification in the aortic rings. These results confirm that Ru360 can directly target vascular tissue and inhibit high-phosphorus-induced vascular calcification.

[0072] Conclusion: This embodiment confirms that in a CKD-related vascular calcification model, MCU expression is significantly upregulated, leading to mitochondrial dynamics imbalance. The MCU-specific inhibitor Ru360 can effectively reduce the degree of vascular calcification and inhibit osteogenic transdifferentiation of VSMCs, and this effect is a direct effect of Ru360 on vascular tissue. These results indicate that Ru360 has a significant therapeutic effect on CKD-related vascular calcification.

[0073] Appendix 1: Primers used for qRT-PCR detection

[0074] Appendix 2: Explanation of Key Terms Mitochondrial calcium uniporter inhibitors are molecules or compounds that can selectively or non-selectively block the function of the mitochondrial calcium uniporter (MCU). The MCU is a calcium ion channel located on the inner mitochondrial membrane, responsible for taking calcium ions from the cytoplasm into the mitochondrial matrix. Inhibiting MCU activity reduces mitochondrial calcium overload, thereby regulating key physiological processes such as cellular energy metabolism, oxidative stress, and apoptosis. Representative drugs include ruthenium red, Ru360, DS16570511, mitoxantrone, KB-R7943, CGP 37157, and berberine.

[0075] BGLAP (osteocalcin): a non-collagenous protein secreted by osteoblasts, used in this invention as a marker for osteogenic transdifferentiation of VSMCs.

[0076] RUNX2: A core transcription factor for osteogenic differentiation, initiating the expression of genes such as BGLAP and ALPL.

[0077] SOX9: a transcription factor involved in cartilage and osteogenic differentiation, upregulated in vascular calcification.

[0078] α-SMA: A marker protein of smooth muscle cell contractile phenotype; its downregulation suggests that VSMCs have lost their contractile function.

[0079] ALPL (alkaline phosphatase): An enzyme involved in calcium phosphate deposition, often used as a late marker of osteogenic differentiation.

[0080] Immunohistochemistry (IHC): Using antibodies to locate and semi-quantitatively analyze specific proteins (such as MCU) in tissue sections.

[0081] qRT-PCR: Real-time quantitative PCR, used to detect the mRNA expression level of genes.

[0082] Western blot: Protein immunoblotting, used to quantitatively detect the expression levels of proteins (such as MCU, BGLAP).

Claims

1. The application of a mitochondrial calcium one-way transporter inhibitor in the preparation of a drug for treating vascular calcification associated with chronic kidney disease, wherein the mitochondrial calcium one-way transporter inhibitor is ruthenium-360.

2. The application according to claim 1, characterized in that: The drug contains a therapeutically effective amount of a mitochondrial calcium oneway transporter inhibitor and a pharmaceutically acceptable carrier.

3. The application according to claim 1, characterized in that: The drug is in the form of an injection.

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