Application of luteolin in treatment of vascular calcification

Abstract

The invention discloses an application of luteolin in treatment of vascular calcification, and relates to the field of biological medicine, and the technical key points are as follows: the luteolin has a synergistic effect to effectively inhibit vascular calcification through a plurality of ways of regulating TOM6, mitochondrial autophagy, NF-kappa B signal pathways and the like; a powerful theoretical basis is provided for further exploring luteolin as treatment intervention, meanwhile, the influence of luteolin on mitochondrial autophagy provides a new perspective for pathophysiology of VC, and a potential treatment direction is provided for VC-related cardiovascular diseases lacking clinical solutions at present.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and particularly to the application of luteolin in the treatment of vascular calcification. Background Art

[0002] Vascular calcification is the process of calcium salt deposition in the blood vessel wall, leading to vascular sclerosis and loss of elasticity, which is commonly seen in diseases such as atherosclerosis, diabetes, and hypertension. Calcification is mainly divided into intimal calcification and adventitial calcification. The former is related to atherosclerosis, while the latter is more common in patients with chronic kidney disease. Vascular calcification increases the risk of cardiovascular diseases, manifested as hardening of the blood vessels and obstruction of blood flow. Common risk factors include age, diabetes, hypertension, chronic kidney disease, and high cholesterol levels. Imaging examinations such as CT and ultrasound can be used for diagnosis. In addition to controlling the underlying diseases, the calcification process can be slowed down through drugs and lifestyle changes.

[0003] Therefore, the present invention aims to provide the application of luteolin in the treatment of vascular calcification to solve the above problems. Summary of the Invention

[0004] The purpose of the present invention is to solve the above problems and provide the application of luteolin in the treatment of vascular calcification. Luteolin effectively inhibits vascular calcification through multiple pathways such as regulating TOM6, mitophagy, and NF-κB signaling pathways.

[0005] To achieve the above purpose, the technical solution of the present invention is as follows:

[0006] The present invention provides the application of luteolin in the treatment of vascular calcification. The luteolin (LU) regulates the calcification of vascular smooth muscle cells (VSMCs) by regulating TOM6, the mitophagy pathway (PINK1 / Parkin), and the NF-κB signaling pathway;

[0007] The luteolin inhibits the osteogenic transformation of vascular smooth muscle cells and inhibits vascular calcification (VC) by downregulating the expression of the osteogenic marker BMP2 and upregulating the expression of the smooth muscle marker α-SMA.

[0008] The luteolin downregulates the expression of the TOM6 gene in vascular smooth muscle cells and inhibits vascular calcification.

[0009] The luteolin promotes mitophagy, clears damaged mitochondria in the patient's body, and inhibits the calcification of vascular smooth muscle cells. In VSMCs, the activation of the PINK1 / Parkin pathway can slow down the calcification process of cells by clearing damaged mitochondria.

[0010] The luteolin inhibits the nuclear translocation of NF-κB, thereby downregulating the expression of TOM6 and slowing down the process of vascular calcification.

[0011] Beneficial effects of this solution compared with the prior art:

[0012] In the present invention, luteolin inhibits the formation of vascular smooth muscle cells (VSMCs) and vascular calcification through multiple mechanisms; firstly, LU inhibits the calcification process of VSMCs by downregulating the osteogenic marker BMP2 and upregulating the smooth muscle marker α-SMA; further, the downregulation of TOM6 can reduce the calcification of VSMCs, and the inhibitory effect of LU is closely related to the expression of TOM6; LU also further slows down vascular calcification by activating the PINK1 / Parkin mitophagy pathway and clearing damaged mitochondria. In addition, LU inhibits the nuclear translocation of NF-κB, reduces the expression of TOM6, and further inhibits the occurrence of vascular calcification. This provides a strong theoretical basis for further exploring luteolin as a therapeutic intervention. At the same time, the effect of luteolin on mitochondrial balance provides a new perspective on the pathophysiology of VC and a potential therapeutic direction for VC-related cardiovascular diseases that currently lack clinical solutions. Description of the Drawings

[0013] Figure 1 It shows that in the examples of the present invention, LU improves the calcification of human vascular smooth muscle cells (VSMCs). Among them, A is the alizarin red staining of calcium deposition in human vascular smooth muscle cells, scale bar = 500 μm; B is the quantitative analysis of alizarin red; C is the determination of calcium content; D is the western blot analysis of a-SMA and BMP2 in human VSMCs; the arrow indicates the calcified lesion, GM = growth medium, CM = calcification medium, LU = luteolin, n = 4;

[0014] Figure 2 It shows that in the examples of the present invention, LU improves the calcification of rat VSMCs. Among them, A is the alizarin red staining of calcium deposition in rat VSMCs, scale bar = 500 μm; B is the quantitative analysis of alizarin red; C is the determination of calcium content; D is the detection of the expression levels of a-SMA and BMP2 in rat VSMCs by Western blotting; the arrow indicates the calcified lesion, GM = growth medium, CM = calcification medium, LU = luteolin, n = 4;

[0015] Figure 3 It shows that in the examples of the present invention, LU improves arterial ring calcification in rat and human models. Among them, A is the alizarin red staining of calcium deposition in the rat arterial ring model, scale bar = 200 μm (upward), scale bar = 500 μm (downward); B is the measurement of calcium content; C is the alizarin red staining of calcium deposition in the human arterial ring model, scale bar = 200 μm (upward), scale bar = 500 μm (downward); D is the measurement of calcium content *p<0.05; GM = growth medium, CM = calcification medium, LU = luteolin, n = 4;

[0016] Figure 4 In the embodiment of the present invention, luteolin (LU) improved aortic calcification in the mouse VC model. Among them, A - B are alizarin red staining of mineral deposition in the mouse aorta and sections; C is the quantitative analysis of calcium content; D - E are western blot analyses of α - SMA and BMP2, and the arrows indicate calcification lesions; vitamin D3 = vitamin D3, LU = luteolin, n = 6;

[0017] Figure 5 In the embodiment of the present invention, RNA - seq revealed the potential mechanism by which LU improves human VSMCs calcification. Among them, A is the volcano plot of different genes in GM vs. CM and CM vs. LU, with red indicating up - regulated genes and blue indicating down - regulated genes; B is the Venn diagram of different genes and their overlap; C is the clustering heatmap of DEGs between samples based on hierarchical clustering, and the red box highlights the six most significantly differentially expressed genes; D is the determination of TOM6, RENBP, NR1H4, and FGF22 mRNA levels in humans and mice by qPCR; E is the western blot analysis of TOM6 protein expression in human VSMCs; F is the immunostaining of TOM6 expression in human VSMCs *p < 0.05, GM = growth medium, CM = calcification medium, LU = luteolin, n = 4;

[0018] Figure 6 In the embodiment of the present invention, inhibiting TOM6 can reduce human VSMC calcification. Among them, A - B are human VSMCs treated with TOM6 - siRNA, stained with alizarin red, and the calcification is quantified; C - D are LU (1 μM) and LV / TOM6 incubated with human VSMCs in the calcification medium, stained with alizarin red, and the calcification is quantified; scale bar = 500 μm *p < 0.05, the arrows indicate calcification lesions, GM = growth medium, CM = calcification medium, LU = luteolin, n = 4;

[0019] Figure 7In the embodiment of the present invention, luteolin (LU) inhibits vascular calcification (VC) by downregulating TOM6 to promote mitophagy. Wherein, A is the immunostaining of the expression of PINK1 and Parkin proteins in human VSMCs; B is the Western blot analysis of the expression of Parkin, PINK1, LC3-II / LC3-I and p62 proteins in human VSMCs; C is the immunostaining of the expression of Parkin and PINK1 proteins on human VSMCs treated with TOM6-siRNA; D-E is that TOM6-siRNA and PINK1 siRNA are used to treat human VSMCs, and then alizarin red staining and quantitative cell calcification are performed; F is the Western blot analysis of the expression of α-SMA and BMP2 proteins in human VSMCs under calcified medium conditions after treatment with TOM6 and PINK1 siRNA. Scale bar = 500 μm, *P<0.05, arrows indicate calcified lesions, GM = growth medium, CM = calcified medium, LU = luteolin, n = 4;

[0020] Figure 8 In the embodiment of the present invention, LU inhibits the nuclear entry of p50, resulting in the downregulation of TOM6 expression. Wherein, A is the prediction of the upstream transcription factor of TOM6 through the UCSC database; B is the immunostaining of the expression of p50 and p65 proteins in human VSMCs; C is the Western blot of the expression of TOM6 protein on human VSMCs after treatment with the NF-κB inhibitor PDTC; D is that human VSMCs are co-transfected with pGL3-basic-pcDNA3.1-3xflag, pGL3-bastic-homo-TOM6-promoter-pcDNA3.1-3xflag, pGL3-basic-p50 + p65, pGL3-bsic-homo-TOM6-promoter + p50 + p65 for 48 hours, and then the firefly and Renilla luciferase activities are measured using the dual luciferase reporter system, the firefly / Renilla luminescence ratio of each well is calculated, and the test sample ratio is normalized to the control ratio. *P<0.05. GM = growth medium, CM = calcified medium, LU = luteolin, NF-κB1 = p50, UCSC database: https: / / genome.ucsc.edu / . n = 4;

[0021] Figure 9 Schematic diagram of LU inhibiting VC in the embodiment of the present invention. LU inhibits the expression of TOM6 by inhibiting the nuclear translocation of NF-κB, thereby promoting autophagy to inhibit VC. Detailed implementation manners

[0022] To enable those skilled in the art to better understand the solution of the present invention, the technical solution of the present invention will be further described in detail below in conjunction with the embodiments and drawings of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the scope of protection of the present invention.

[0023] It should be noted that, without conflict, the embodiments in the present invention and the features in the embodiments can be combined with each other. The present invention will be described in detail below in conjunction with the embodiments.

[0024] Embodiment:

[0025] 1. Cell culture

[0026] Rat aortic VSMCs were obtained from 8-week-old male Sprague-Dawley (SD) rats (Animal Care Center of Southern Medical University, China). After death, the thoracic aorta was carefully dissected, and then the media was excised and cut into small pieces. Then these fragments were cultured in a CO2 incubator at 37°C. After VSMCs migrated from the segments, the medium was renewed. Human VSMCs from the American Type Culture Collection were cultured in a complete growth medium supplemented with 10% fetal bovine serum (FBS) at 37°C and 5% CO2. VSMCs of passages 3-8 were used in all experiments to maintain the consistency and reliability of the results. VSMCs were cultured in 35-mm culture dishes until 80% confluence, and then they were exposed to a high-phosphate and calcium medium (10 mmol / L β-glycerophosphate and 3 mmol / L CaCl2) or a medium containing a specific drug. The experimental design included three groups: control medium (GM), calcification medium (CM), and CM plus LU (0.2 μM, 0.5 μM, and 1 μM). Each in vitro test was independently repeated four times.

[0027] 2. Organ culture of aortic rings

[0028] Eight-week-old male SD rats were used. The thoracic aorta of the rats was aseptically isolated and cut into vascular rings approximately 5 mm in length. Subsequently, these rings were cultured in a growth medium in a humidified incubator at 37°C. Ethically obtained from amputated human arterial tissues, approved by the Ethics Committee of Zhujiang Hospital, Southern Medical University, China (2021-KY-079-02) in accordance with the principles outlined in the Declaration of Helsinki. All patients provided written informed consent. The vascular rings were cultured in GM, CM, or LU for 7 days, and the medium was renewed every 48 hours. Subsequently, samples were collected for subsequent analysis. The experiments involving aortic rings were independently conducted four times.

[0029] 3. Animal studies

[0030] All animal experiments in this study were conducted in accordance with the Guide for the Care and Use of Laboratory Animals (National Research Council, USA) and approved by the Animal Experiment Ethics Committee of Zhujiang Hospital, Southern Medical University, China (approval number: LAEC-2024-110). As previously described, the mouse aortic calcification model was induced by vitamin D3 (VitD3). Male C57BL / 6 mice were randomly divided into three groups: control group (Ctrl, n = 6), model group receiving vitamin D3 (VitD3, n = 6), and LU model group (vitamin D3 + LU, n = 6). Mice in the vitamin D3 and vitamin D3 + LU groups were subcutaneously injected with vitamin D3 at a dose of 5×10 5 IU / kg for three consecutive days. The VitD3 + LU group received an intraperitoneal injection of LU at a dose of 20 mg / kg. After the experiment was completed, the animals were euthanized, and their aortas were collected for further analysis.

[0031] 4. Alizarin red staining

[0032] Alizarin red staining was used to evaluate calcification in VSMCs. Rat VSMCs were fixed in 4% paraformaldehyde for 10 minutes, washed with phosphate-buffered saline (PBS), and then stained with 2% alizarin red solution for 5 minutes at ambient temperature. After removing the excess dye with deionized water, calcium deposition was observed using a Leica Microsystems microscope. The degree of calcification was determined by measuring the absorbance at 405 nm after eluting the alizarin red dye with 10% formic acid. Subsequently, the sections were dewaxed, stained with 2% alizarin red solution for 5 minutes, and observed using an inverted microscope. In the case of whole-aorta staining, the dissected aorta was first fixed in 95% ethanol for 24 hours, then stained overnight with 0.003% alizarin red solution in 1% potassium hydroxide. Then the aorta samples were rinsed with 2% potassium hydroxide before imaging. After alizarin red staining, formic acid was added to the cells, and the resulting solution was transferred to a 96-well plate for absorbance measurement at 405 nm to evaluate the differences between experimental groups.

[0033] 5. Calcium content determination

[0034] Calcium levels were quantified using a calcium assay kit from Leagene Biotechnology Co., Ltd., Beijing, China, strictly according to the protocol outlined by the manufacturer. Samples were treated with MTB solution, incubated, and then their absorbance was read at 610 nm. The BCA method was used to evaluate protein concentration, and the calcium content was expressed as μg / mg protein.

[0035] 6. RNA sequencing

[0036] Human VSMCs were divided into three different groups: CM, GM, and LU. Total RNA was isolated from each group using triazole. Subsequently, three separate RNA-seq libraries were generated and sequenced. The clean reads obtained for each group were aligned, and transcriptome assembly was performed separately. Differential expression analysis was carried out to identify the differentially expressed genes (DEGs) among the CM, GM, and LU groups. The next-generation sequencing data related to the present invention have been deposited in NCBI (BioProject PRJNA1121653).

[0037] 7. Western blot analysis

[0038] Protein samples derived from cells and aortic tissues were separated by polyacrylamide gel electrophoresis and then transferred onto PVDF membranes. Subsequently, the membranes were incubated at 4°C with antibodies targeting BMP2 (1:1,000, Abcam, USA, ab214821), α-SMA (1:1,000), TOM6 (1:1,000; Proteintech, USA, 16689-1-AP), PINK1 (1:1,000, Proteintech, USA, 23274-1-AP, CST, USA, 6956s), and β-actin (1:1,000, Proteintech, USA, HRP-60008). After incubation with the primary antibodies, the membranes were washed three times with TBST and then incubated with the secondary antibodies diluted in TBST for 2 hours. Subsequently, the membranes were washed three more times. Detection of protein bands was achieved using ECL reagent, and the resulting data were saved for analysis. Then, the gray values of the protein bands were evaluated using Image J software to determine any differences.

[0039] 8. Quantitative real-time PCR

[0040] Total RNA was extracted using TRIzol reagent (Thermo Fisher Scientific, USA), and 1 μg of each RNA sample was reverse-transcribed using TaKaRa PrimeScript TM RT Master Mix. The target genes were amplified using an Applied Biosystems real-time fluorescence quantitative PCR instrument, with β-actin as the internal standard. The polymerase chain reaction (PCR) primers used are shown in Table 2. Based on the corresponding Ct values obtained, the gene expression levels were calculated using the 2-ΔΔCt method.

[0041] 9. Immunocytofluorescence

[0042] The cells were fixed with 4% paraformaldehyde solution, permeabilized with cell permeabilization buffer, and blocked with goat blocking serum. Subsequently, the primary antibodies (TOM6, p50) were incubated overnight, and then the fluorescent secondary antibody (goat anti-rabbit IgG) was incubated. Finally, the nuclei were stained, and the selected areas were imaged using an inverted fluorescence microscope.

[0043] 10. Small interfering RNA (siRNA) transfection

[0044] Human VSMCs were grown in 35-mm culture dishes until 80% confluence was reached. Next, according to the provided guidelines, cells were transfected with TOM6 and PINK1 siRNAs (from Ribobio, Guangzhou, China) at a final concentration of 30 nM using Lipofectamine 3000 (Thermo Fisher, Waltham, MA, USA). The expression levels of TOM6 and PINK1 in VSMCs transfected with these siRNAs were detected using Western blotting.

[0045] 11. Lentiviral infection

[0046] To generate stable expression of TOM6 in cells, lentiviruses were produced by co-transfecting HEK293T cells with the transfer plasmid pLvx-CMV-TOM6-P2A-mNeonGreen-WPRE or pLvx-CMV mNeonGree WPRE (negative control) and the packaging plasmids pMD2.G and psPAX2. After 72 hours, the lentiviral supernatant was collected and filtered. VSMCs were seeded in 6-well plates at a density of 2 x 105 cells per well to reach a confluence rate of approximately 75% the next day and were transduced with the lentiviral supernatant in the presence of 8 μg / mL polybrene. After 24 hours, the medium was changed. Seven days after transduction, the expression of mNeonGreen was analyzed using FACS to confirm the transduction efficiency. Positive cells were sorted to establish a stable cell line.

[0047] 12. Luciferase assay

[0048] Human VSMCs were cultured in 24-well plates, and at the indicated time intervals (24, 48, 72, and 96 hours), 20 μl of the medium was taken from three separate wells and transferred to a 96-well microplate for luciferase activity assessment. The QUANTI-Luc detection kit was used according to the manufacturer's guidelines. As indicated, a microplate reader was used to measure luciferase activity, which was set to 50 μl injection, endpoint measurement, 4-second initial pause, and 0.5-second read time.

[0049] 13. Statistical analysis

[0050] All quantitative data are presented as the mean ± standard deviation (SD). Statistical analysis was performed using GraphPad Prism version 8.0. For experiments comparing two groups, Student's t-test was used to evaluate statistical significance. In cases involving multiple groups, one-way analysis of variance (ANOVA) was performed to determine whether there were statistically significant differences between the means of three or more independent groups. When ANOVA yielded significant results, Tukey's multiple comparison test was subsequently performed as a post hoc analysis. A p-value less than 0.05 was considered statistically significant.

[0051] 14. Results

[0052] 14.1 LU inhibits calcification of VSMCs

[0053] To investigate the potential beneficial effects of LU on calcification of rat and human VSMCs, rat VSMCs and human VSMCs were intervened with the same concentrations of LU (0.2 μM, 0.5 μM, 1 μM) for 7 days under CM conditions. Alizarin red staining showed that CM significantly induced VSMCs calcification. LU dose-dependently reduced VSMCs calcification. In addition, quantitative analysis of alizarin red staining indicated that LU significantly decreased calcium deposition in VSMCs ( Figure 1 A - B, Figure 2 A - B, p < 0.05). Consistent with the results of alizarin red staining, LU significantly decreased the calcium content in VSMCs ( Figure 1 C and 2C, p < 0.05). In addition, western blot analysis showed that LU significantly decreased the expression level of the osteogenic marker BMP2 and increased the expression of α-SMA ( Figure 1 D and 2D). These data indicate that LU can inhibit VSMCs calcification, and a concentration of 1 μM can most effectively reduce cell calcification.

[0054] 14.2 LU inhibits calcification of isolated arterial rings

[0055] In human and rat samples, the calcification medium induced arterial ring calcification. Then, the effects of LU on arterial calcification in human and rat vascular tissues were investigated. Hematoxylin and eosin staining of paraffin sections showed that LU significantly inhibited arterial ring calcification and decreased the calcium content in arterial rings compared with the calcification group ( Figure 3 A and 3C), which was supported by the quantification of calcium content ( Figure 3 B and 3D). These results further confirm that LU can inhibit VC induced by high calcium and high phosphorus.

[0056] 14.3 LU inhibits vitamin D3-induced arterial calcification in mice

[0057] To evaluate the therapeutic effect of LU on VC in vivo, 20 mg / kg of LU was administered to VitD3-induced calcified mice over 10 days. The mice were randomly divided into three groups: control group, vitamin D3 group, and vitamin D3 + LU group. Compared with the VitD3 group, alizarin red staining analysis of the whole aorta and aortic sections of LU-treated mice showed reduced calcium deposition ( Figure 4 A-B), which was supported by the quantification of calcium content ( Figure 4 C). In addition, western blot analysis showed that LU treatment led to decreased BMP2 expression and increased a-SMA expression ( Figure 4 D-E).

[0058] 14.4 Analysis of differential gene expression of LU inhibiting VC by RNA-seq

[0059] To clarify the mechanism of LU inhibiting VC, the RNA-seq profiles of the GM group and the CM group were compared, and 330 DEGs were detected in the CM group ( Figure 5 A). Similarly, comparing the RNA-seq profiles of the CM group and the LU group, 318 DEGs were detected in the LU group ( Figure 5 A). To identify the genes that might play a key role in the inhibitory effect of LU on VC, the commonalities between these two groups of DEGs were found. By this method, 75 overlapping genes were identified ( Figure 5 B). Among these genes, six genes showing the most significant differential expression were selected for further study: TNFRSF6B, UPK3BL1, TOM6, RENBP, NR1H4, and FGF22 ( Figure 5C). Notably, four of these genes, including TOM6, RENBP, NR1H4, and FGF22, have a high degree of homology between humans and mice, indicating their potential roles in conserved biological processes. After carefully considering the characteristics of other candidate genes, TOM6 was focused on in subsequent experiments. TNFRSF6B and UPK3BL1 were excluded because of their low homology between humans and mice, which may limit the translatability of mouse model findings to human biology. Although RENBP, NR1H4, and FGF22 show a high degree of homology between species, their roles in vascular calcification have been relatively well studied compared to TOM6. RENBP is involved in the biosynthesis of N-acetylneuraminic acid, and its clear function in sialic acid biosynthesis may make it more difficult to establish a direct link between RENBP and the pathogenesis of vascular calcification compared to other candidate genes with less prominent roles in cellular processes. The nuclear receptor NR1H4 has been reported to play a role in cholesterol and bile acid metabolism, which may affect vascular health. FGF22 is a member of the fibroblast growth factor family and is related to angiogenesis and endothelial cell function. In contrast, the literature on TOM6 in the context of vascular calcification is limited, providing new insights into revealing its role in this pathological process. To validate the RNA-seq findings, further qPCR analysis was performed on human and mouse VSMCs. The expression of TOM6 was significantly upregulated in calcified VSMCs, while LU significantly downregulated the expression of TOM6 ( Figure 5 D). WB and immunofluorescence analyses confirmed this finding, indicating that LU significantly reduced the expression level of TOM6 in human VSMCs ( Figure 5 E-F). These results together suggest that TOM6 may play a key role in LU-mediated VC inhibition.

[0060] 14.5 LU reduces HVSMCs calcification by inhibiting TOM6

[0061] To further investigate the effect of TOM6 on VC, we transfected human VSMCs with TOM6 siRNA and lentivirus encoding TOM6 (LV / TOM6). Alizarin red staining showed that TOM6-siRNA inhibited the calcification of VSMCs when exposed to calcifying medium ( Figure 6 A), and LV / TOM6 promoted calcification in VSMCs (see Figure 6 C). Then, we investigated the potential role of TOM6 in mediating the inhibitory effect of LU on VSMC calcification. The staining results showed that overexpression of TOM6 using lentivirus promoted calcification and counteracted the inhibitory effect of LU in human VSMCs treated with calcifying medium ( Figure 6 C). Quantitative evaluation of alizarin red staining confirmed these results (Figure 6 B and D). Therefore, these results suggest that TOM6 signaling may play a role in mediating the inhibitory effect of LU on VSMC calcification.

[0062] 14.6 LU promotes mitophagy to inhibit VC by downregulating TOM6

[0063] Previous studies have shown that mitophagy is associated with VC and LU promotes mitophagy. To investigate how LU inhibits VC, its effect on mitophagy was studied. The PINK1 / Parkin pathway was examined and protein levels were measured. Immunofluorescence analysis showed that the expression of PINK1 and Parkin was decreased in the calcification group compared with the control group, while the expression of PINK1 / Parkin was significantly increased in the LU treatment group ( Figure 7 A). Subsequently, the western blot results confirmed these findings, showing an increased LC3-II / LC3-I ratio and a decreased p62 expression in the LU treatment group, indicating that LU may induce mitophagy, thereby alleviating VC ( Figure 7 B). Subsequently, it was investigated whether LU regulates mitophagy through its effect on TOM6, thereby affecting VC. Treatment with TOM6 siRNA inhibited the decrease in PINK1 and Parkin in calcified VSMCs ( Figure 7 C). In addition, the results of alizarin red staining and quantitative analysis showed that the combined use of TOM6-siRNA and PINK1-siRNA counteracted the promoting effect of calcified VSMCs on VC compared with the use of TOM6-siRNA alone ( Figure 7 D-E). In addition, western blot analysis showed that the combined treatment with TOM6 siRNA and PINK1 siRNA could counteract the upregulation of BMP2 expression and the downregulation of α-SMA expression in calcified VSMCs ( Figure 7 F). The TOM6 / PINK1 signaling pathway may be involved in the inhibitory mechanism of LU on the osteogenic differentiation and calcification of VSMCs.

[0064] 14.7 LU downregulates the expression of TOM6 by inhibiting the nuclear entry of NF-κB

[0065] The regulatory mechanism of LU in regulating TOM6 gene expression was studied. Through analysis with the UCSC database, NF-κB1 (p50) was identified as an upstream transcription factor of TOM6 and was significantly correlated with the expression of TOM6 ( Figure 8 A). Subsequently, an attempt was made to explore whether LU mediates the inhibitory effect of TOM6 on vascular smooth muscle cell calcification through NF-κB. Immunofluorescence results showed that LU could inhibit the entry of p50 and p65 into the nucleus ( Figure 8 B). In addition, a significant decrease in TOM6 expression was observed after treating human VSMCs with the NF-κB inhibitor PDTCFigure 8 C), which further confirmed the crucial role of NF-κB in regulating TOM6 expression. The results of luciferase reporter gene assays showed that p50 and p65 could significantly enhance the activity of the TOM6 promoter region ( Figure 8 D). In summary, LU may downregulate the expression of TOM6 by inhibiting the nuclear entry of NF-κB, thereby affecting the calcification process of VSMCs.

[0066] 15. Conclusions

[0067] By binding to the NF-κB / TOM6 / PINK1 signaling pathway, it promoted mitophagy and successfully inhibited the progression of VC. The consistent positive results from in vitro and in vivo analyses provided a strong theoretical basis for further exploring luteolin as a therapeutic intervention. In addition, the effect of luteolin on mitochondrial homeostasis provided a new perspective on the pathophysiology of VC and a potential therapeutic direction for VC-related cardiovascular diseases for which there is currently a lack of clinical solutions.

[0068] The above specific embodiments are merely explanations of the present invention and are not limitations thereof. Those skilled in the art may make modifications to the embodiments without creative contributions according to needs after reading this specification, but as long as they are within the scope of the claims of the present invention, they are protected by the patent law.

Claims

1. Use of luteolin in the treatment of vascular calcification, characterized in that: The luteolin regulates the calcification of vascular smooth muscle cells by modulating TOM6, mitophagy, and the NF-κB signaling pathway; The luteolin inhibits the osteogenic transformation of vascular smooth muscle cells and vascular calcification by downregulating the expression of the osteogenic marker BMP2 and upregulating the expression of the smooth muscle marker α-SMA.

2. The use of luteolin in the treatment of vascular calcification according to claim 1, characterized in that: The luteolin downregulates the expression of the TOM6 gene in vascular smooth muscle cells, thereby inhibiting vascular calcification.

3. The application of luteolin in the treatment of vascular calcification according to claim 1, characterized in that: The luteolin promotes mitophagy, clears damaged mitochondria in the patient's body, and inhibits the calcification of vascular smooth muscle cells.

4. The application of luteolin in the treatment of vascular calcification according to claim 2, characterized in that: The luteolin inhibits the nuclear translocation of NF-κB, thereby downregulating the expression of TOM6 and slowing down the process of vascular calcification.

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