Application of apigenin to improvement of hypertension vascular remodeling
Through the effects of apilobin and TP53, the proliferation and migration of vascular smooth muscle cells induced by Ang II are inhibited, and the problem of limited improvement of existing hypertensive treatments on vascular remodeling is solved, and the significant inhibitory effect on hypertensive vascular remodeling is achieved.
Patent Information
- Application Number
- CN202510452885.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-11
- Publication Date
- 2025-06-24
AI Technical Summary
The existing hypertensive treatment methods have limited effect on improving vascular remodeling, and have side effects, and lack effective treatment methods to block or reverse vascular remodeling in the early stage.
By studying the effects of apilobin, it was found that it can act with TP53, inhibiting the proliferation and migration of vascular smooth muscle cells induced by Ang II, thereby reducing vascular remodeling caused by hypertension.
Apilobin significantly inhibited vascular remodeling in hypertensive rats, reduced the degree of aortic fibrosis, and verified its protective effect on improving hypertensive vascular remodeling through in vivo and intra-vitro experiments.
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Figure CN120189406A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of pharmaceutical technology, and in particular to the application of apigenin in improving hypertensive vascular remodeling. Background Art
[0002] Hypertension has become one of the important risk factors for cardiovascular diseases. Long-term hypertension can lead to changes in the structure and function of blood vessels, namely vascular remodeling. Vascular remodeling is the pathophysiological basis for the deterioration and target organ damage of hypertension. The structural manifestations of hypertensive vascular remodeling are: thickening of the blood vessel wall, narrowing of the lumen, and an increase in the ratio of the thickness of the vascular media to the inner diameter of the lumen. Vascular stenosis can lead to reduced tissue blood supply and, in severe cases, damage the functions of important target organs such as the heart, brain, and kidneys. Vascular remodeling is the result of the interaction of multiple factors. Among them, abnormal proliferation and migration of smooth muscle cells are the central link of hypertensive vascular remodeling. Vascular smooth muscle cells are the main components of the blood vessel wall and have high plasticity. Under normal physiological conditions, they maintain a differentiated state to maintain the structural integrity and physiological function of the blood vessel wall; however, under the stimulation of various pathological factors such as oxidized low-density lipoprotein, angiotensin II, and platelet-derived growth factor, they can undergo abnormal proliferation and migration.
[0003] The treatment of hypertensive vascular diseases is mainly based on the following aspects: First, strengthen the self-management of patients and carry out regular and moderate exercise under the guidance of a doctor; second, pay attention to diet in daily life and reduce the intake of sodium salts; in terms of drug treatment, the currently recognized main drugs include four categories: angiotensin II (Ang II) receptor antagonists, β-blockers, Ca 2+ antagonists, and diuretics. However, the drugs for improving hypertension have a very limited effect on improving the vascular remodeling caused by hypertension, and at the same time, there are side effects to varying degrees, such as dizziness, headache, gastrointestinal reactions, and kidney function damage. With the in-depth research on Chinese herbal medicines, more natural compounds provide alternative therapies for the treatment of hypertension and its complications. Therefore, seeking key therapeutic drugs and targets and early blocking or reversing vascular remodeling are key issues in the prevention and treatment of hypertension.
[0004] Apigenin (4′,5,7-trihydroxyflavone) is a flavonoid compound widely distributed in vegetables and fruits in warm and temperate zones, with the highest content in celery. The protective effect and mechanism of apigenin in hypertensive vascular remodeling have not been reported yet. Summary of the Invention
[0005] The technical problem to be solved by the present invention is to provide the application of apigenin in improving hypertensive vascular remodeling, deeply explore the protective effect and mechanism of apigenin on vascular remodeling caused by hypertension, and clarify the role of apigenin in improving hypertensive vascular remodeling.
[0006] To solve the above technical problems, the present invention adopts the following technical solutions:
[0007] Application of apigenin in improving hypertensive vascular remodeling.
[0008] Preferably, apigenin acts on TP53, and TP53 is the key target for apigenin to improve hypertensive vascular remodeling.
[0009] Preferably, apigenin inhibits Ang II-induced VSMCs proliferation by activating TP53.
[0010] Preferably, apigenin inhibits Ang II-induced VSMCs migration by activating TP53.
[0011] Preferably, apigenin reduces vascular remodeling in hypertensive rats by upregulating the expression of TP53.
[0012] Application of apigenin in the preparation of a drug for improving hypertensive vascular remodeling.
[0013] Advantages of the present invention:
[0014] The research of the present invention deeply explores the protective effect and mechanism of apigenin on vascular remodeling caused by hypertension, clarifies the role of apigenin in improving hypertensive vascular remodeling, and uses network pharmacology and a variety of technical means to find that TP53 is the key target for apigenin to improve hypertensive vascular remodeling, providing a theoretical basis for finding new therapeutic drugs for the prevention and treatment of vascular remodeling caused by hypertension.
[0015] Through in vivo experiments using the SHR spontaneous hypertensive rat model, the results of the present invention show that: apigenin can significantly inhibit the increase in wall thickness in the vascular cross-section of SHR rats, reduce the degree of aortic fibrosis, and thus improve vascular remodeling caused by hypertension.
[0016] Through in vitro experiments using an Ang II-induced vascular smooth muscle cell (VSMCs) model, the results show that: apigenin can inhibit the proliferation and migration of Ang II-induced VSMCs in a concentration-dependent manner to improve hypertensive vascular remodeling.
[0017] The present invention also verifies through network pharmacology, molecular docking and in vivo and in vitro experiments that activating TP53 is the key for apigenin to improve hypertensive vascular remodeling. Description of the drawings
[0018] Figure 1 It is a schematic flow chart for exploring the effect of apigenin on aortic vascular remodeling in hypertensive rats at the in vivo level of the present invention;
[0019] Figure 2Schematic flow chart for exploring the effect of apigenin on the proliferation and migration of vascular smooth muscle cells induced by Ang II at the in vitro level of the present invention;
[0020] Figure 3 Schematic flow chart for the network pharmacology of the present invention;
[0021] Figure 4 Schematic flow chart for exploring whether the TP53 inhibitor can block the protective effect of apigenin on the proliferation and migration of smooth muscle cells induced by Ang-II in the in vitro experiment of the present invention;
[0022] Figure 5 Experimental results of apigenin inhibiting aortic vascular remodeling in SHR rats of the present invention;
[0023] Figure 6 Experimental results of apigenin inhibiting the proliferation of smooth muscle cells induced by Ang II of the present invention;
[0024] Figure 7 Experimental results of apigenin inhibiting the proliferation of smooth muscle cells induced by Ang II through G0 / G1 phase cell arrest of the present invention;
[0025] Figure 8 Experimental results of apigenin inhibiting the migration of smooth muscle cells induced by Ang II of the present invention;
[0026] Figure 9 Network pharmacology for exploring the mechanism of action of apigenin of the present invention;
[0027] Figure 10 Experimental results of apigenin inhibiting the proliferation and migration of VSMCs induced by Ang II through multiple targets of VEGFA, TP53 and AKT1 of the present invention;
[0028] Figure 11 Experimental results of apigenin inhibiting the proliferation of VSMCs induced by Ang II by activating TP53 of the present invention.
[0029] Figure 12 Experimental results of apigenin inhibiting the migration of VSMCs induced by Ang II by activating TP53 of the present invention
[0030] Figure 13 Experimental results of apigenin improving vascular remodeling in hypertensive rats by upregulating TP53 of the present invention. Specific embodiments
[0031] For the convenience of those skilled in the art, the present invention will be further described below in conjunction with the embodiments and the accompanying drawings. The content mentioned in the embodiments does not limit the present invention.
[0032] Example 1
[0033] Effect of Apigenin on Vascular Remodeling in SHR Hypertensive Rats and Proliferation and Migration of Rat Smooth Muscle Cells Induced by AngⅡ
[0034] (1) In vivo experiment:
[0035] Male 7-week-old SHR hypertensive rats and WKY rats (body weight 150 g - 170 g) were selected. Sixteen SHR rats were randomly divided into a control group (gavaged with CMC-Na) and an apigenin group (gavaged with 50 mg / kg); sixteen WKY rats were divided in the same way as above. Blood pressure of the animals was measured every two weeks after administration. After 8 weeks of administration, the thoracic aorta was taken for subsequent experiments. HE staining was used to observe the morphology, wall thickness and wall-lumen ratio of blood vessels, Masson staining was used to observe the fibrosis of blood vessels, and Western blot was used to detect the expression of PCNA, MMP2 / 9, proteins related to the proliferation and migration of vascular smooth muscle.
[0036] (2) In vitro experiment
[0037] Primary thoracic aortic smooth muscle cells (VSMCs), passages 4 - 7, were used in all cell experiments. The experiments were divided into five groups: normal group, model group (1 μM Ang-II), and on the basis of the model, apigenin (10, 12.5 and 15 μM) was given for intervention for 24 h. CCK8 colorimetric method was used to detect cell viability, EDU method was used to detect cell proliferation, scratch test and Transwell test were used to detect cell proliferation, flow cytometry was used to detect cell cycle, and Western blot method was used to detect the expression of PCNA, MMP2 / 9, TP53 proteins related to cell proliferation and migration, and CDK4 / 6, CycD1 proteins related to cell cycle.
[0038] Apigenin inhibits vascular remodeling in SHR rats:
[0039] During the 10-week gavage of rats, compared with the CMC-Na group, there were no abnormal manifestations in the general conditions such as diet, water intake, urine volume, etc. of the rats in the apigenin gavage group (SHR + apigenin gavage group and WKY + apigenin gavage group). The changes in blood pressure and body weight of rats in each group were monitored as follows: There was no statistical significance in the comparison of body weight of rats in each time period (P > 0.05); compared with the SHR group, in the SHR + apigenin gavage group of rats, the systolic blood pressure decreased significantly 4 weeks after administration ( ## P < 0.01), and the decrease in systolic blood pressure tended to be stable at 8 weeks of administration ( ### P < 0.001). The results of H&E staining showed that the aortic vascular parameters of the SHR group, including wall thickness, lumen diameter and their ratio, were increased compared with the WKY group, while these indicators in the SHR group treated with apigenin were decreased ( Figure 5c-f). Masson staining results showed that compared with the WKY group, aortic cross-sectional fibrosis in SHR rats increased. After treatment with apigenin, the degree of fibrosis decreased ( Figure 5 g). Immunohistochemical analysis and Western blot results showed that apigenin had a certain inhibitory effect on the protein expression of proliferation-related index PCNA and migration-related index MMP2 ( Figure 5 h and i).
[0040] Apigenin inhibits Ang II-induced proliferation and migration of vascular smooth muscle cells:
[0041] Vascular smooth muscle cells (VSMCs) induced by Ang II were treated with different concentrations of apigenin (0, 10, 12.5, and 15 μM), and the results were as Figure 6 shown in a-b. Treatment with different concentrations of apigenin (0, 10, 12.5, and 15 μM) could inhibit cell proliferation induced by Ang II and the increase in PCNA protein expression ( Figure 6 d-e). At the same time, flow cytometry was used to analyze the cell cycle, and the results showed that apigenin inhibited Ang II-induced VSMCs proliferation by increasing the G0 / G1 phase ( Figure 7 ).
[0042] The scratch assay and TranswellTM assay were used to detect the migration ability of cells in each group. The results showed that compared with the Ang II group, treatment with apigenin could inhibit the lateral and longitudinal migration of VSMCs induced by Ang II. At the same time, apigenin could concentration-dependently reduce the expression of MMP2 and MMP9 ( Figure 8 ).
[0043] Example 2
[0044] Network pharmacology combined with molecular docking to predict the mechanism of action of apigenin in improving hypertension-induced vascular remodeling:
[0045] The research of this invention uses the TCMSP (http: / / tcmspw.com / tcmsp.php) database, the SWISS Targetprediction database (http: / / swisstargetprediction.ch), the SEA database: (http: / / sea.bkslab.org), and the QSAR (quantitative structure activity relationships) database to predict the potential targets of apigenin. The genecards database (https: / / www.genecards.org), the OMIM database (http: / / omim.org), and the diegenet database (http: / / www.disgenet.org) are used to predict the potential targets of hypertensive vascular remodeling. The abbreviations of all target genes are put into the Unitprot database (https: / / www.Unitprot.org) to be converted into gene uniport IDs. After removing their respective duplicates, they are the potential targets for the drug to exert its effects and the potential targets of the disease. All the targets of the two are put into the Omicshare database (https: / / www.omicshare.com) to make a Venn diagram to find the common targets at www.ensembl.org / biomart. The common targets are put into the String database (https: / / string-db.org) to obtain the results of protein-protein interaction between the two. The results of protein-protein interaction between the two are put into the Cytoscape software to find the Hub gene and the possible KEGG and GO pathways that may play a role. Molecular docking uses DiscoveryStudio TM 2.5 performs computer-aided molecular docking.
[0046] The Swiss Target Prediction database, the SEA database, and the QSAR database are used to predict the action targets of apigenin. After removing duplicate targets, a total of 270 targets are obtained. The GeneCards database, the OMIM database, and the Disgenet database are used to predict the targets of hypertensive vascular remodeling disease. After removing duplicates, a total of 1160 targets are obtained. The results of these two predictions are put into the Omicshare database, and Venn analysis yields 103 common targets, such as Figure 9 shown in a. The 103 common targets are put into the String database to obtain the results of protein-protein interaction. The protein-protein interaction is put into the Cytoscape software to find the top 20 key genes, such as Figure 9As shown in Figure b, the top 5 key targets are AKT1, TP53, INS, VEGFA, and Caspase 3. Among these targets, AKT1, TP53, and VEGFA are related to migration and proliferation. The results of KEGG enrichment analysis showed that the PI3K-AKT pathway and the TP53 pathway were significantly enriched ( Figure 9 c). The results of GO enrichment analysis showed that the cell proliferation process was significantly enriched ( Figure 9 d). The results of molecular docking showed that the LibDock score of apigenin docked with AKT1 was 90.808, and the LibDock score of the AKT1 inhibitor (A674563) was 116.046; the LibDock score of apigenin and TP53 was 81.3166, and the LibDock score of the TP53 activator (RITA 3) was 71.6861; the LibDock score of apigenin and VEGFA docked was 95.1967, and the LibDock score of the VEGFA inhibitor was 101.141( Figure 10 a-c). Western blot was used to verify the prediction of molecular docking, and the results showed that apigenin could regulate the expression of p-AKT1, TP53, and VEGFA( Figure 10 d-i).
[0047] Taken together, the above results suggest that apigenin may inhibit the proliferation and migration of AngⅡ-induced VSMCs by regulating multiple targets such as VEGFA, TP53, and AKT1.
[0048] Example 3
[0049] In vitro and in vivo experiments were conducted to verify whether apigenin improves hypertensive vascular remodeling by activating the TP53 target:
[0050] (1) In vivo experiment
[0051] Male 7-week-old SHR hypertensive rats and WKY rats (body weight 150g - 170g) were selected. 16 SHR rats were randomly divided into a control group (gavaged with CMC-Na) and an apigenin group (gavaged with 50mg / kg); 16 WKY rats were divided in the same way as above. The blood pressure of the animals was measured every two weeks after administration. After 8 weeks of administration, the thoracic aorta was taken for subsequent experiments. Immunohistochemistry and Western blot were used to detect the expression and localization of the vascular smooth muscle proliferation and migration-related protein TP53. The results of Western blot and immunohistochemistry showed that compared with the WKY group, the expression of TP53 in the SHR group decreased, and the expression of TP53 in the SHR group increased after apigenin administration, while there was no obvious change in the WKY group( Figure 13 a-c).
[0052] (2) In vitro experiment
[0053] Molecular docking results showed that, compared with the activator, apigenin showed a higher docking score, suggesting that apigenin might improve hypertensive vascular remodeling by directly acting on the P53 target. To further verify whether apigenin inhibits Ang-II-induced VSMCs proliferation by activating the TP53 target, VSMCs were pretreated with the TP53 inhibitor PFT-α (10 μM) for 1 hour, and then Ang-II and apigenin (12.5 μM) were added for co-treatment for 48 hours. Cell viability was detected by CCK8 colorimetric assay (APEXBIO), cell proliferation was detected by EDU assay, and cell proliferation was detected by scratch assay and Transwell assay. Western blot and qRT-PCR were used to detect the expression of cell proliferation and migration-related proteins PCNA, MMP2 / 9, and TP53.
[0054] Molecular docking results showed that the docking score of TP53 with apigenin was higher than that of the TP53 activator (RITA 3). It was speculated that TP53 might play a key role in apigenin-inhibited AngⅡ-induced vascular proliferation and migration. The TP53-specific inhibitor PFT-α was pretreated in vascular smooth muscle incubated with AngⅡ. The results showed that PFT-α could inhibit the upregulation of TP53 expression by apigenin ( Figure 11 b).
[0055] VSMCs were pretreated with the TP53 inhibitor PFT-α (10 μM) for 1 h, and then AngⅡ and apigenin (12.5 μM) were added for co-treatment for 48 h. The results of the EdU assay for detecting cell proliferation were as Figure 11 shown in a-c: PFT-α (TP53 inhibitor) inhibited the anti-proliferative effect of apigenin on AngⅡ-induced VSMCs, and the expression of PCNA increased compared with the apigenin group ( Figure 11 d). And apigenin could reduce the cell cycle proteins CDK4 / 6 and Cyclin D1, inhibiting cell proliferation. After treatment with PFT-α, the expression levels of the above three proteins increased again, promoting cell proliferation ( Figure 11 d). The results indicated that apigenin inhibited the proliferation of VSMCs by blocking the cell cycle in the G0 / G1 phase.
[0056] The results of the cell scratch assay and transwell migration assay showed that: after pretreatment with PFT-α, the scratch healing area increased significantly ( Figure 12 a and Figure 12 b). In addition, compared with the apigenin group, the expression level of MMP2 protein increased after pretreatment with PFT-α ( Figure 12 e). These results indicated that TP53 might be a key target of apigenin in AngⅡ-induced smooth muscle cell proliferation and migration.
[0057] In summary, these results indicate that apigenin reduces vascular remodeling in hypertensive rats by upregulating the expression of TP53.
[0058] All technical features in this embodiment can be modified in appearance according to actual needs.
[0059] The above embodiments are preferred implementation schemes of the present invention. In addition, the present invention can also be implemented in other ways. Any obvious substitution without departing from the concept of the technical solution of the present invention is within the protection scope of the present invention.
Claims
1. Application of apigenin in improving vascular remodeling in hypertension.
2. The use of apigenin in improving hypertensive vascular remodeling according to claim 1, characterized in that: Apigenin interacts with TP53, which is the key target of apigenin in improving vascular remodeling in hypertension.
3. The use of apigenin in improving hypertensive vascular remodeling according to claim 2, characterized in that: Apigenin inhibits Ang II-induced VSMCs proliferation by activating TP53.
4. The use of apigenin in improving hypertensive vascular remodeling according to claim 2, characterized in that: Apigenin inhibits Ang II-induced VSMCs migration by activating TP53.
5. The use of apigenin in improving hypertensive vascular remodeling according to claim 2, characterized in that: Apigenin reduces vascular remodeling in hypertensive rats by upregulating TP53 expression.
6. Application of apigenin in the preparation of drugs for improving vascular remodeling in hypertension.