Application of quasi-kinesization modification inhibitor in preparation of medicine for preventing and / or treating chronic kidney disease vascular calcification

Abstract

The invention discloses application of a quasi-kinesization modification inhibitor in preparation of a medicine for preventing and / or treating vascular calcification of chronic kidney diseases. The micafungin serving as a metalloid modification inhibitor inhibits osteogenic transdifferentiation of vascular smooth muscle cells, reduces the expression of an osteogenic factor RUNX2 and increases the expression of a contraction marker alpha-SMA of the vascular smooth muscle cells. Meanwhile, calcium salt deposition of vascular smooth muscle cells is remarkably inhibited. It is found for the first time that micafungin can play a role in protecting CKD vascular calcification independent of the antifungal effect of micafungin. The discovery provides a new hope for prevention and treatment of CKD vascular calcification.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of medicine, and particularly relates to the application of mizoribine in the preparation of a drug for preventing and / or treating vascular calcification in chronic kidney disease. BACKGROUND

[0002] The global incidence of chronic kidney disease (CKD) is 10% to 15%, and about 5 million to 10 million people die every year, which has become a prominent global public health problem. On May 23, 2025, the 78th World Health Assembly listed kidney disease as one of the major non-communicable diseases that should be given priority attention globally. Although the quality of treatment and management of CKD patients has been greatly improved in recent years, the life expectancy of CKD patients at all stages is still significantly reduced. Vascular calcification is one of the important culprits of death in patients with chronic kidney disease. Vascular calcification is the deposition of calcium-phosphorus crystal minerals on the blood vessel wall, and calcification can be found in all arteries of the body and can be located in the intima and / or media of the artery. Intimal calcification usually occurs in the background of atherosclerosis and is often associated with traditional atherosclerotic risk factors. However, medial calcification is more characteristic in CKD patients and can occur without the influence of additional metabolic factors. Medial calcification in CKD patients is a multi-factor involved and strictly regulated active process, which has many similarities with embryonic osteogenesis, and its process involves a variety of interrelated molecular mechanisms. Vascular smooth muscle cells (VSMCs) are the main cells involved in CKD vascular calcification, and osteogenic transdifferentiation of VSMCs is a key link in the occurrence of vascular calcification under the environment of high phosphorus, uremic toxins and other factors in CKD patients. At present, there is still a lack of effective intervention and treatment methods for CKD vascular calcification. Based on the serious disease burden caused by CKD vascular calcification, exploring effective therapies for CKD vascular calcification is a key clinical problem that needs to be solved urgently.

[0003] Micafungin belongs to the echinocandin class of broad-spectrum antifungal drugs, which can inhibit the synthesis of 1,3-beta-D-glucan, destroy the fungal cell wall, and change the osmotic pressure inside and outside the fungal cell, thereby dissolving and dying. Micafungin was approved by the US Food and Drug Administration (FDA) in 2005, and was approved for listing in China in 2007, and was approved for use in the European Union in 2008. It is currently used clinically to treat and prevent invasive fungal infections, including fungemia, respiratory fungal disease, and gastrointestinal fungal disease. Micafungin is administered intravenously, and meets the linear pharmacokinetics, without a first loading dose, and can reach an effective blood drug concentration on the day of administration, is mainly excreted through feces and bile, and a small part is excreted through the kidney. The results of clinical studies show that among similar products, micafungin has good safety, does not need to adjust the dose for patients with liver and kidney dysfunction, children and the elderly, and can be used with immunosuppressants and other drugs, rarely has drug interactions, and has a relatively low price. There is no public report that micafungin can treat chronic kidney disease vascular calcification. SUMMARY

[0004] The technical problem solved by the present application is to provide the application of the mimetic modification inhibitor in the preparation of a drug for preventing and / or treating chronic kidney disease vascular calcification.

[0005] In a specific embodiment, the mimetic modification inhibitor is micafungin.

[0006] The vascular calcification is medial calcification.

[0007] The present application finds that micafungin inhibits the osteogenic transdifferentiation of vascular smooth muscle cells induced by high-phosphorus stimulation, reduces the expression of the osteogenic factor RUNX2, and increases the expression of the contraction marker alpha-SMA of vascular smooth muscle cells. At the same time, it significantly inhibits the calcium deposition of vascular smooth muscle cells. In a CKD vascular calcification model mouse, micafungin significantly relieves vascular calcification.

[0008] Beneficial effects: The present application first finds that micafungin can independently of its antifungal effect play a protective role on CKD vascular calcification. This finding provides new hope for the prevention and treatment of CKD vascular calcification. At the same time, micafungin, as a widely used clinical drug, is used for the treatment of CKD vascular calcification with a new mechanism, greatly reducing the time and economic cost of new drugs entering the clinic, and has great clinical transformation value. BRIEF DESCRIPTION OF DRAWINGS

[0009] Figure 1Figure showing that blocking pseudomodification can reduce vascular calcification in CKD mice. A shows the effect of blocking pseudomodification on Runx2 and a-SMA mRNA levels; B and C show representative Western Blot images and statistical analysis of the effect of blocking pseudomodification on RUNX2 and a-SMA protein levels; D shows representative images of alizarin red staining of the effect of blocking pseudomodification on VSMC calcium deposition; E shows the effect of blocking pseudomodification on VSMC calcium content; G shows the experimental procedure for constructing a CKD vascular calcification model mouse and using tail vein injection of adeno-associated virus (AAV) to knock down UBA3; H and I show representative Western Blot images and statistical analysis of the efficiency of AAV knockdown of UBA3; J shows the body weight results of the mice in each group; K shows the serum creatinine detection results of the mice in each group; L shows the blood urea nitrogen detection results of the mice in each group; M shows the blood inorganic phosphorus detection results of the mice in each group; N shows PAS (top) and Masson staining (bottom) of the kidney tissues of the mice in each group; O and P show representative Western Blot images and statistical analysis of the protein levels of RUNX2 and a-SMA in the arterial tissues of the mice in each group; Q shows the general alizarin red staining of the aortic calcium deposition of the mice in each group; R shows representative images of alizarin red staining (top) and Von Kossa staining (bottom) of the aortic tissue calcium deposition of the mice in each group; (Figure legend AP model: adenine combined with high-phosphorus diet model; GM+siNC: complete growth medium+empty siRNA; GM+siUBA3: complete growth medium+UBA3 knockdown; CM+siNC: calcification medium+empty siRNA; CM+siUBA3: calcification medium+UBA3 knockdown; CTL+AAV-NC: healthy control+empty AAV; CTL+AAV-shUBA3: healthy control+UBA3 knockdown; CKD+AAV-NC: CKD+empty AAV; CKD+AAV-shUBA3: CKD+UBA3 knockdown); Figure 2Figure 9 is a result graph of micafeinum inhibiting high-phosphorus-induced osteogenic transdifferentiation and calcium deposition of vascular smooth muscle cells, wherein A is a representative picture of alizarin red staining of the effect of different concentrations of micafeinum (5 μM, 10 μM, 20 μM) on calcium deposition of VSMCs; B and C are a representative picture of Western Blot and statistical analysis of the effect of different concentrations of micafeinum (5 μM, 10 μM, 20 μM) intervention on the protein level of VSMCs RUNX2 and α-SMA; D is the effect of micafeinum (20 μM) on the level of ossification modification; E is the effect of micafeinum (20 μM) on the mRNA level of VSMCs Runx2 and α-SMA; F is the effect of micafeinum (20 μM) on the calcium content of VSMCs; G and H are a representative picture of Western Blot and statistical analysis of the effect of micafeinum (20 μM) on the protein level of VSMCs RUNX2 and α-SMA; I is a representative picture of immunofluorescence staining of the effect of micafeinum (20 μM) on the expression level of VSMCs RUNX2; J is a representative picture of alizarin red staining of the effect of micafeinum (20 μM) on calcium deposition of VSMCs. (Note: GM: complete growth medium; CM: calcification medium containing high-phosphorus; CM+Mica: calcification medium + micafeinum intervention). Figure 3 Figure 10 is a result graph of micafeinum reducing vascular calcification of CKD mice, wherein A is the animal modeling and micafeinum intervention process, B is PAS (top) and Masson staining (bottom) of the kidney tissue of mice in each group; C is the body weight result of mice in each group; D is the detection result of serum creatinine of mice in each group; E is the detection result of blood urea nitrogen of mice in each group; F is the detection result of blood phosphorus of mice in each group; G and H are a representative picture of Western Blot and statistical analysis of the protein expression level of RUNX2 and α-SMA of the arterial tissue of mice in each group, I is a representative picture of immunohistochemical staining of the effect of micafeinum on the vascular RUNX2 expression level of CKD mice; J is a representative picture of immunofluorescence co-staining of RUNX2 and α-SMA of the arterial tissue of mice in each group; K is a representative picture of gross alizarin red staining of the effect of micafeinum on the calcium deposition of the arterial tissue of CKD mice; L is a representative picture of alizarin red and Von Kossa staining of the effect of micafeinum on the calcium deposition of the arterial tissue of CKD mice. (Note: AP model: adenine combined with high-phosphorus diet model; CTL: healthy control group; CKD+Saline: CKD+normal saline injection group; CKD+Mica: CKD+micafeinum injection group; Scr: serum creatinine; BUN: blood urea nitrogen; Pi: serum inorganic phosphorus). DETAILED DESCRIPTION

[0010] The above and / or other aspects of the present application will become more apparent by describing in detail the preferred embodiments thereof with reference to the attached drawings, wherein:

[0011] The experimental procedures and experimental conditions involved in the following examples are as follows: 1. Cell culture: (1) The aortic tissue of 4-week-old male C57BL / 6J mice was taken. The adventitia and intima of the aortic tissue were carefully removed, and the tissue was cut into 1 mm size tissue pieces, collagenase II (Sigma, USA) and elastase (Worthingtonbiomedical, USA) solution was added, and the tissue was incubated in a 5% CO2, 37°C constant temperature cell incubator for 1 hour. The cell suspension was centrifuged and resuspended in Dulbecco's modified Eagle's medium / nutrient mixture F-12 medium (DMEM / F12) (Gibco, USA) supplemented with 20% fetal bovine serum (FBS) (Wisent, Canada) and 1X penicillin-streptomycin (KEYGEN, China). The cells were seeded in a cell culture flask (Corning, USA) and incubated in a 5% carbon dioxide, 37°C cell incubator. The culture medium was changed every 2 days, and the cells were subcultured when the cell density reached 80% ~ 90%. VSMCs of passage 3 ~ 6 were used for experiments.

[0012] (2) Preparation of cell culture medium: ① Complete growth medium: DMEM / F12 medium + 1X penicillin-streptomycin + 10% or 20% FBS ② Control growth medium (GM): DMEM / F12 medium + 1X penicillin-streptomycin + 5% FBS; ③ Calcification medium (CM): DMEM / F12 medium + 1X penicillin-streptomycin + 5% FBS + 10mM β-glycerophosphate + 50ug / ml ascorbic acid.

[0013] (3) Establishment and intervention of CKD vascular calcification model in vitro: When the cell density of VSMCs seeded on the cell culture plate after subculture reached 70%-80%, the complete growth medium was replaced with 10% complete growth medium without antibiotics, and siRNA was transfected using Lipo8000 (Beyotime, China). Dilute siRNA and Lipo8000 in Opti-MEM (Gibco, USA) and mix well, incubate at room temperature for 20 minutes. Add the mixture to the cell culture medium. After 6-8 hours, replace the corresponding medium according to the experimental conditions. The siUBA3 sequence used is: UBA3 siRNA Sense 5'-GAGAGAGCAUCACAAAUAUAAU-3', UBA3 siRNA Anti-sense 5'-UAUAUUGUGAUGCUCUCUCUA-3'.

[0014] VSMCs were inoculated on cell culture plates when the cell density reached 60%-70% after passage. The complete growth medium was replaced with GM or CM culture. When the density of VSMCs in CM culture reached 80%, mica or an equal volume of DMSO was added. The medium and intervention reagent were replaced every two days, and the culture was carried out for 7 days. According to the different treatment conditions, the cells were divided into GM group, CM+DMSO group, CM+Mica (different concentrations) group.

[0015] 2、Animal model: The CKD vascular calcification model mice were established by a modified adenine combined with high-phosphorus diet induction method. To avoid the interference of estrogen on vascular calcification, we used 8-week-old male C57 / BL6J mice for the experiment.

[0016] For animal experiments by knocking down UBA3 to block sumoylation, 9-type adeno-associated virus (AAV) specific to vascular smooth muscle cells UBA3 was injected through the tail vein with an SM22a promoter. After 3 weeks of injection, AAV stably expressed, and the CKD vascular calcification model was established. The mice were randomly divided into healthy control+AAV empty group (CTL+AAV-NC), healthy control+AAV knockdown UBA3 group (CTL+AAV-shUBA3 group), CKD+AAV empty (CKD+AAV-NC), and CKD+AAV knockdown UBA3 group (CKD+AAV-shUBA3).

[0017] For animal experiments of mica intervention, the mice were randomly divided into healthy control group (CTL), CKD vascular calcification mice combined with normal saline injection group (CKD+Saline), and CKD vascular calcification mice combined with mica injection group (CKD+Mica). The CKD vascular calcification model mice were constructed by feeding with 0.2% adenine and 1.8% high-phosphorus feed for 12 weeks. The healthy mice were fed with regular diet for 12 weeks. The CKD mice were given 10 mg / kg mica or an equal volume of normal saline subcutaneously every day. After feeding, euthanasia was performed by inhaling isoflurane, and serum and kidney tissue were collected. The mouse aortic arch to bilateral iliac artery was stripped to obtain the aortic tissue.

[0018] 3、Western Blot experiment Proteins were extracted using RIPA buffer (FDbio, China) supplemented with protease and phosphatase inhibitors. Protein concentration was determined using BCA protein assay kit (KEYGEN, China). Proteins were separated by SDS-PAGE and transferred to 0.22 pm PVDF membranes (Roche, UK). Membranes were blocked with 5% skim milk for 1 hour at room temperature, and after TBST washes, membranes were incubated with primary antibodies overnight at 4°C. After washing, membranes were incubated with HRP-conjugated secondary antibodies (ZSQB, China) for 1 hour at room temperature. Blots were visualized with chemiluminescent reagent (FDbio, China) and analyzed using ImageJ software. Data were expressed as fold change relative to control group.

[0019] 4. Quantitative real-time polymerase chain reaction (qRT-PCR) Total RNA was isolated using FastPure Cell / Tissue Total RNA Isolation Kit (Vazyme, China) and cDNA was synthesized using Reverse Transcription Kit (Vazyme, China) according to the manufacturer’s instructions. Real-time quantitative polymerase chain reaction (qRT-PCR) was performed using SYBR qPCR Master Mix (Vazyme, China) on a Roche real-time PCR system. The relative expression of each gene was normalized to the expression of Gapdh and analyzed using the 2^ -AACt method.

[0020] 5. Alizarin red staining (1) Cell alizarin red staining: Cell culture medium was removed and cells were gently washed with PBS buffer for 3 times. 4% paraformaldehyde solution was added and cells were fixed at room temperature for 15 minutes. After removing the paraformaldehyde, cells were gently washed with PBS for 3 times. Appropriate amount of 2% alizarin red staining (Oricell, USA) was added according to the size of the culture dish and incubated at room temperature for 10 minutes in the dark. After stopping the staining, cells were gently washed with double distilled water for 3 times. The distribution of calcium nodules was recorded by camera or microscopic imaging system.

[0021] (2) Gross alizarin red staining: After aortic tissue was fixed with paraformaldehyde for 24 hours, it was dehydrated with 95% ethanol for 12 hours and then immersed in a solution containing 0.003% alizarin red dye and 1% potassium hydroxide for 1 hour in the dark. After staining was completed, the background staining was removed by washing with 2% potassium hydroxide solution twice until the blood vessels appeared translucent.

[0022] 6. Calcium content determination Calcium ion content in VSMCs was detected using a calcium ion detection kit (Nanjing Jiancheng, China). The procedure was performed according to the instructions of the kit. Briefly, cells were collected, washed with PBS, resuspended in deionized water, and sonicated to lyse the cells. After centrifugation, the supernatant was collected and reacted with the color reagent provided in the kit. The calcium concentration was determined by measuring the absorbance at 610 nm using a microplate reader. The total calcium content was normalized to the total protein concentration determined using the BCA assay.

[0023] 7. Immunofluorescence staining Paraffin-embedded vascular tissue sections were cut into 5-μm-thick sections and rehydrated after deparaffinization. Antigen retrieval was performed by heat mediation using Tris / EDTA buffer (pH 9.0) or sodium citrate buffer (pH 6.0). The sections were blocked with blocking buffer (Beyotime, China) at room temperature for 1 h and then incubated with primary antibodies at 4°C overnight. The next day, the sections were incubated with Alexa Fluor 488 (Abways, China) or Alexa Fluor 594 (Abways, China) at room temperature for 1 h in the dark. The nuclei were counterstained with DAPI (KEYGEN, China) at room temperature for 8 min. The sections were mounted using an anti-quenching mounting medium (Beyotime, China) and imaged using a Leica Microsystems (DM4000B-1).

[0024] VSMCs were grown on sterile glass coverslips in 24-well plates. After the end of the intervention, the cells were washed three times with PBS, fixed in 4% paraformaldehyde at room temperature for 15 min, and washed three times with PBS. Permeabilization was performed using 0.1% Triton X-100 for 10 min. The cells were then incubated with blocking buffer at room temperature for 1 h and then incubated with primary antibodies at 4°C overnight. The primary antibodies were coupled with fluorescently labeled secondary antibodies (Abways, China) at room temperature for 1 h in the dark. The nuclei were stained with DAPI (KEYGEN, China) at room temperature for 8 min in the dark. Finally, the sections were mounted using an anti-quenching fluorescent mounting medium, and images were acquired using a Leica Microsystems (DM4000B-1).

[0025] 8. Immunohistochemical staining Immunohistochemistry (IHC) staining was performed using 5 pm thick paraffin-embedded tissue sections. Sections were deparaffinized and rehydrated. Depending on the primary antibody, heat-mediated antigen retrieval was performed using Tris / EDTA buffer, pH 9.0, or sodium citrate buffer, pH 6.0. Sections were incubated with blocking buffer (Beyotime, China) for 1 hour at room temperature to reduce non-specific binding, followed by incubation with primary antibody overnight at 4 °C, and then incubation with secondary antibody (MXB, China) for 1 hour at room temperature. Color development was performed using DAB chromogen (MXB, China), followed by hematoxylin (KGYGEN, China) staining. Images were taken using a microscope (DM4000B-1, Leica, Germany).

[0026] 9. Serum creatinine, urea nitrogen, and blood phosphorus detection Serum creatinine and blood phosphorus were detected using a Nanjing Jiancheng (China) kit according to the instructions. Urea nitrogen was detected using a Abbott (China) kit according to the instructions.

[0027] 10. Kidney tissue PAS, Masson staining Paraffin-embedded kidney tissue was cut into 3 pm thick sections and deparaffinized. PAS staining: sections were incubated with 0.5% periodic acid solution for 10 minutes, washed, and then stained with Schiff reagent for 15 minutes (Solarbio, China). After re-staining with hematoxylin, drying, and mounting. Masson staining was performed according to the standard protocol using Weigert hematoxylin iron, Biebrich scarlet-acid fuchsin, and aniline blue for staining.

[0028] Example 1 Effect of blocking neddylation modification on CKD vascular calcification.

[0029] Neddylation modification requires the cascade catalysis of NEDD8 activating enzyme E1, NEDD8 binding enzyme E2, and NEDD8 ligase E3. The only E1 enzyme of neddylation modification is a heterodimer composed of catalytic subunit UBA3 and regulatory subunit NAE1. Knocking down UBA3 can block the occurrence of neddylation from the source.

[0030] (1) Cell experiment In vitro, mouse VSMCs were cultured and induced to osteogenic transdifferentiation by high-phosphate stimulation to mimic vascular calcification in CKD. Small interfering RNA (siRNA) was used to knock down UBA3 in VSMCs to block sumoylation modification. Cell calcification was observed. qRT-PCR and WB showed that, at both mRNA and protein levels, blocking sumoylation modification reduced the expression of VSMC osteogenic factor RUNX2 induced by high-phosphate stimulation and increased the expression of vascular smooth muscle cell contraction marker a-SMA (Fig. 1A-D). Using alizarin red staining and cell calcium content determination to measure VSMC calcium salt deposition, it was found that blocking sumoylation modification could significantly reduce the calcium salt deposition of VSMCs induced by high-phosphate (Fig. 1E and Fig. 1F). Figure 1 E and Fig. 1F).

[0031] (2) Animal experiments Animal experiments were performed using 8-week-old male C57BL / 6J mice. CKD vascular calcification model mice were constructed by feeding adenine combined with high-phosphate diet for 12 weeks; UBA3 in vascular smooth muscle cells was specifically knocked down by injecting adeno-associated virus (AAV) through the tail vein (i.v). Three weeks after injection of AAV, AAV stable expression began, and the adenine combined with high-phosphate diet modeling began. Mice were randomly divided into four groups: healthy control mice + injection of empty AAV group (CTL + AAV-NC), healthy control mice + injection of UBA3 knockdown AAV group (CTL + AAV-shUBA3), CKD mice + injection of empty AAV group (CKD + AAV-NC), and CKD mice + injection of UBA3 knockdown AAV group (CKD + AAV-shUBA3). The mouse modeling and intervention process is shown in Fig. 1G. After the modeling was completed, the body weight of the mice was measured, and the mice were euthanized using isoflurane, and the eyeball blood, kidney tissue and aortic tissue were taken for subsequent experiments. Serum creatinine, urea nitrogen, and blood phosphorus were detected to assess kidney damage and modeling. The results showed that the CKD model mice were successfully modeled and blocking sumoylation modification slightly alleviated the weight loss of CKD mice, but had no significant improvement in serum creatinine, urea nitrogen, blood phosphorus, and renal histological damage (Fig. 1J-N). This indicates that blocking sumoylation modification has no protective effect on kidney function. Western Blot experiment to detect AAV efficiency, the results showed that UBA3 knockdown was successful (Fig. 1H and Fig. 1I). Figure 1 H and Figure 1 I). Western Blot experiment to detect the expression of RUNX2 and a-SMA proteins in the aortic tissue of mice, blocking sumoylation modification significantly reduced the expression of RUNX2 in the aortic tissue of CKD mice and increased the expression of a-SMA protein, with statistically significant differences (Fig. 1H and Fig. 1I). Figure 1O and FIG. 1P). Masson's trichrome staining of arterial tissue reflects the calcium salt deposition, and the results show that blocking neddylation modification significantly reduces the calcium salt deposition in blood vessels of CKD mice (FIG. 1Q). Masson's trichrome staining and Von Kossa staining of arterial tissue sections reflect the degree of vascular calcification, and the results show that blocking neddylation modification significantly reduces vascular calcification in CKD mice (FIG. 1R). Figure 1 R}.

[0032] The above in vivo and in vitro experimental results show that blocking neddylation modification can significantly reduce CKD vascular calcification.

[0033] Example 2 Effect of Mivafungin on osteogenic transdifferentiation of VSMCs

[0034] (1) Cell experiment Mouse VSMCs were cultured in vitro, and osteogenic transdifferentiation of VSMCs was induced by high-phosphorus stimulation to simulate CKD vascular calcification. Different concentrations of mivafungin (5 μM, 10 μM, 20 μM) or the same volume of DMSO were used for intervention, and the calcification of cells was observed. Alizarin red staining was used to observe the calcium salt deposition of VSMCs. Alizarin red staining showed that mivafungin inhibited the calcium salt deposition of VSMCs caused by high-phosphorus stimulation in a dose-dependent manner (FIG. 2A). Figure 2 A). Western Blot analysis showed that mivafungin could reduce the expression of VSMCs RUNX2 induced by high-phosphorus stimulation in a dose-dependent manner, and increase the expression of α-SMA (FIG. 2B, Figure 2 B, Figure 2 C).

[0035] Previous experiments showed that 20 μM of mivafungin had the strongest inhibitory effect on osteogenic transdifferentiation of VSMCs without obvious cytotoxicity. Therefore, 20 μM of mivafungin was selected for subsequent experiments. First, we detected the effect of mivafungin on the global neddylation modification level of VSMCs. Cell proteins were extracted from the GM group, the CM+DMSO group, and the CM+Mica group, and the NEDD8 expression in each group was detected by Western Blot. The results showed that compared with the GM group, the NEDD8 level in the CM+DMSO group was significantly increased, and mivafungin intervention (CM+Mica) could significantly reduce the overall NEDD8 level. This indicates that in calcified VSMCs, mivafungin can significantly inhibit the global neddylation modification level (FIG. 2D). Figure 2

[0036] Next, the effect of 20 μM mivafungin on osteogenic transdifferentiation and calcium salt deposition of VSMCs was further verified. qRT-PCR experiments showed that compared with the CM+DMSO group, the mivafungin intervention group (CM+Mica) significantly reduced the mRNA level of Runx2 and increased the mRNA level of α-SMA (FIG. 2E). Figure 2 ​E). Western Blot experiment showed that, compared with the CM+DMSO group, the RUNX2 protein level of the Micafungin intervention group (CM+Mica) was significantly decreased, and the a-SMA protein level was significantly increased, and the difference was statistically significant (P<0.05) Figure 2 G, Figure 2 H). Immunofluorescence staining of the cell osteogenic marker RUNX2 showed that, compared with the calcification group, the Micafungin intervention group could reduce the expression of RUNX2 (P<0.05) Figure 2 I). Alizarin red staining experiment and cell calcium content determination showed that Micafungin intervention significantly reduced the calcium salt deposition of VSMCs under CM culture (P<0.05) Figure 2 J、 Figure 2 F). The above results all showed that Micafungin could significantly inhibit the osteogenic transdifferentiation and calcium salt deposition of VSMCs induced by high-phosphorus stimulation.

[0037] (2) Animal experiment.

[0038] To further verify the alleviating effect of Micafungin on CKD vascular calcification, a CKD vascular calcification model mouse was constructed and given Micafungin intervention for in vivo experiment.

[0039] 8-week-old male C57BL / 6J mice were used for animal experiment. CKD vascular calcification model mice were constructed by feeding with adenine combined with high-phosphorus diet for 12 weeks; Micafungin 10 mg / kg or the same volume of normal saline was subcutaneously injected once a day to intervene the mice. The mice were randomly divided into the following groups: 1. Healthy control group (CTL); 2. CKD+normal saline intervention group (CKD+Saline); 3. CKD+Micafungin intervention group (CKD+Mica). The modeling and intervention process of the mice is shown in Figure 3 A. After the modeling was completed, the body weight of the mice was measured, and the mice were euthanized with isoflurane, and the eyeball blood, kidney tissue and aortic tissue were taken for subsequent experiments. PAS and Masson staining was performed on the kidney tissue of each group of mice, and compared with the CTL group, the kidney structure of the CKD+Saline and CKD+Mica mice was extensively damaged, and showed significant fibrosis (P<0.05) Figure 3 B). The body weight of each group of mice was found to be significantly reduced in the CKD mice compared with the CTL group, and Micafungin intervention did not significantly increase the body weight of the CKD mice (P>0.05) Figure 3 C). The serum creatinine, urea nitrogen and blood phosphorus of the mice were detected, and compared with the CTL group, the serum creatinine, urea nitrogen and inorganic phosphorus levels of the CKD group were significantly increased, and Micafungin intervention did not reduce the serum creatinine, urea nitrogen and inorganic phosphorus levels (P>0.05) Figure 3 D、 Figure 3 E、 Figure 3F). The above results show that the CKD mouse model is successfully constructed, and mica intervention does not improve the renal function of CKD mice. Next, we use mouse aortic tissue to perform Western Blot, alizarin red staining and immunohistochemical staining to observe the vascular calcification of mice in each group. Western Blot experiment shows that compared with the CTL group, the RUNX2 protein level of CKD+Saline mice is significantly increased, and the α-SMA protein level is significantly reduced, and mica intervention (CKD+Mica) reverses this pro-calcification effect, that is, mica intervention significantly reduces the expression of RUNX2 in the aortic tissue of CKD mice, and increases the expression of α-SMA Figure 3 G、 Figure 3 H). In addition, immunohistochemical and immunofluorescence staining of osteogenic factor RUNX2 on aortic tissue shows that mica intervention significantly reduces the expression of RUNX2 in the blood vessels of CKD mice Figure 3 I and Figure 3 J). The aortic tissue was subjected to gross alizarin red staining, and the results showed that mica significantly alleviated the vascular calcification of CKD mice Figure 3 K). Alizarin red staining and Von Kossa staining of paraffin sections of aortic tissue also verified the same conclusion, that is, mica significantly alleviated the vascular calcification of CKD mice ​ L) The above results show that mica can significantly alleviate the vascular calcification of CKD mice, and this protective effect is independent of the effect on renal function.

[0040] In summary, we first found that mica can independently of its antifungal effect, play a protective role in CKD vascular calcification by inhibiting the pseudo-modification, which provides new hope for the prevention and treatment of CKD vascular calcification. At the same time, mica, as a widely used clinical drug, is innovatively used for the treatment of CKD vascular calcification with a new mechanism, greatly reducing the time and economic cost of new drugs entering the clinic, and has great clinical transformation value.

[0041] The present application provides a protective effect of mica on CKD vascular calcification, and there are many methods and ways to achieve this technical solution. The above description is only the preferred embodiment of the present application, and it should be pointed out that for ordinary skilled persons in the technical field, without departing from the principle of the present application, some improvements and refinements can be made, which should be regarded as the protection scope of the present application. The components not explicitly described in the embodiment can be realized by existing technology.

Claims

1. Application of mimetic modification inhibitors in the preparation of drugs for the prevention and / or treatment of vascular calcification in chronic kidney disease.

2. The application according to claim 1, characterized in that, The mimetic modification inhibitor is micafungin.

3. The application according to claim 1, characterized in that, The vascular calcification is medial calcification.

4. The application according to claim 2, characterized in that, Micafungin inhibits osteogenic transdifferentiation of vascular smooth muscle cells.

5. The application according to claim 2, characterized in that, Micafenoxan reduces the expression of osteogenic factor RUNX2.

6. The application according to claim 2, characterized in that, Micafungin increases the expression of α-SMA, a marker of vascular smooth muscle cell contraction.

7. The application according to claim 2, characterized in that, Micafungin significantly inhibits calcium salt deposition in vascular smooth muscle cells.

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