Use of klf2 agonists in the manufacture of a medicament for reducing aortic aging

Abstract

The application belongs to the field of cardiovascular disease treatment, and provides application of a KLF2 agonist in preparation of a drug for reducing aortic aging. In the research of the application, the activation of the KLF2 signal pathway by Juglanin regulates the aging-related phenotype conversion process of vascular smooth muscle cells in the aging aortic wall, inhibits abnormal extracellular matrix secretion, and promotes the maintenance of the contractile phenotype function, thereby improving the pathological remodeling state of the aortic wall and maintaining the structural stability of the aortic wall, and solving the problem that in the prior art, there is a lack of targeted molecular intervention means for aortic aging.

Description

Technical Field

[0001] This application pertains to the field of cardiovascular disease treatment, specifically, it provides the use of KLF2 agonists in the preparation of drugs to reduce aortic aging. Background Technology

[0002] With the accelerating aging of the population, the incidence of age-related vascular diseases is increasing year by year. Among them, aortic aging is considered one of the important pathological bases for many serious cardiovascular events. During aortic aging, the vessel wall undergoes significant changes at both the structural and functional levels, mainly manifested as the breakage and reduction of elastic fibers, abnormal deposition of extracellular matrix such as collagen, decreased vascular compliance, and imbalance in the functional state of vascular smooth muscle cells. These changes gradually transform the aorta from a highly elastic vessel into a rigid structure, weakening its buffering capacity for pulsatile blood flow, thereby increasing the burden on the cardiovascular system and raising the risk of aortic dilation, rupture, and dissection.

[0003] Under long-term aging and hemodynamic stress, the aforementioned structural degeneration can further lead to local dilation of the aortic wall and decreased wall stability, eventually developing into an aortic aneurysm. This disease is characterized by local or diffuse irreversible dilation of the aorta, and is characterized by high progression, high risk of rupture, and poor prognosis, making it one of the most serious fatal cardiovascular diseases in the elderly population.

[0004] In the context of aging, dysfunction of vascular smooth muscle cells is considered a key factor driving aortic structural degeneration and decreased mechanical properties. Existing research indicates that aging can induce age-related phenotypic shifts in vascular smooth muscle cells, characterized by loss of contractile phenotype, increased extracellular matrix secretion, and activation of inflammation-related phenotypes, thereby disrupting vascular wall structural stability and promoting pathological remodeling. However, the molecular regulatory mechanisms of this process are not fully elucidated, and effective drugs that can directly intervene in age-related phenotypic shifts of vascular smooth muscle cells are lacking in clinical practice.

[0005] Krüppel-like factor 2 (KLF2) is an important transcriptional regulator that has been shown to play a crucial role in maintaining vascular cell homeostasis. Previous studies have suggested that the KLF2 signaling pathway can participate in regulating age-related phenotypic transitions in vascular smooth muscle cells, thereby helping to stabilize vascular wall structure and improve its mechanical properties by inhibiting abnormal extracellular matrix secretion and maintaining or restoring the expression of contractile phenotypic genes. However, in age-related aortic lesions, the activation mechanism of the KLF2 signaling pathway and its pharmacological regulation still lack clear technical solutions.

[0006] Juglanin is a naturally derived small molecule compound with reported biological activities, but current technologies mainly focus on its effects in inflammation, oxidative stress, or other non-vascular-related diseases. Whether juglanin can affect aging-related phenotypic transitions, extracellular matrix secretion, and contractile function of vascular smooth muscle cells by regulating the KLF2 signaling pathway remains unclear, lacking systematic research and well-defined technological applications.

[0007] Current drug interventions for aortic aging or age-related aortic diseases primarily focus on blood pressure control or non-specific anti-inflammatory and anti-fibrotic therapies, failing to precisely intervene at the molecular level in addressing aging-related phenotypic abnormalities of vascular smooth muscle cells and degeneration of vascular wall structure. Therefore, providing a novel technical solution to activate the KLF2 signaling pathway, regulate aging-related phenotypic transitions of vascular smooth muscle cells, thereby improving aortic structural and functional degeneration and inhibiting the occurrence or progression of age-related aortic aneurysms remains a pressing technical challenge in this field. Summary of the Invention

[0008] The purpose of this invention is to provide the application of KLF2 agonists in the preparation of drugs to alleviate aortic aging, such as the application in drugs for treating or preventing aortic dilatation and for treating or preventing aortic aging-related aneurysms, in order to solve the problems in the prior art of lacking effective drug intervention for aortic aging-related diseases and being unable to intervene in the phenotypic transformation of vascular smooth muscle cells and pathological remodeling of the vascular wall at the molecular level.

[0009] On the one hand, this application provides the use of KLF2 agonists in the preparation of drugs to alleviate aortic aging.

[0010] Furthermore, the drug provides one or more of the following effects: (1) Inhibit age-related molecular phenotypic abnormalities and functional imbalances in vascular smooth muscle cells; (2) Regulate the abnormal secretion and deposition of extracellular matrix, and reduce pathological remodeling of the aortic wall; (3) Promote the expression of genes related to vascular smooth muscle cell contraction and maintain or restore the integrity of the aortic media structure.

[0011] Furthermore, the aging-related molecular phenotypic abnormalities are the upregulation of p16 and / or p21 expression.

[0012] Furthermore, regulating the abnormal secretion and deposition of the extracellular matrix includes reducing FN1 expression.

[0013] Furthermore, the vascular smooth muscle cell contraction-related genes are α-SMA and / or SM22α.

[0014] On the other hand, this application provides the use of KLF2 agonists in the preparation of medicaments for the treatment or prevention of aortic dilation.

[0015] On the other hand, this application provides the use of KLF2 agonists in the preparation of medicaments for the treatment or prevention of aortic aging-related aneurysms.

[0016] Furthermore, the KLF2 agonist is Juglanin.

[0017] Furthermore, Juglanin is the sole active ingredient in the drug.

[0018] Furthermore, the drug is in oral or injectable form.

[0019] Furthermore, the drug is an oral dosage form.

[0020] Furthermore, the drug also contains pharmaceutically acceptable excipients.

[0021] Those skilled in the art can select and design available dosage forms based on pharmaceutical common sense, including but not limited to the oral and injectable dosage forms mentioned above. Excipients that can be used in the above dosage forms include solvents, cosolvents, antioxidants, suspending agents, pH adjusters, osmotic pressure adjusters, fillers, paste bases, lubricants, dispersants, coatings, capsule shells, sustained-release agents, and controlled-release agents.

[0022] The juglanin described in this application is also known as jujube glycoside, chemically named kaempferol 3-O-alpha-L-arabinofuranoside, CAS number 5041-67-8, and its structure is shown in Formula I below:

[0023] Formula I Beneficial effects: This invention regulates the aging-related phenotypic transition of vascular smooth muscle cells in the aging aortic wall by activating the KLF2 signaling pathway with juglanin, inhibits abnormal extracellular matrix secretion, and promotes the maintenance of contractile phenotype function, thereby improving the pathological remodeling state of the aortic wall and maintaining its structural stability. Compared with the prior art, this invention has at least the following technical effects: It can intervene at the molecular level in the key pathological basis of aortic aging—the imbalance of vascular smooth muscle cell function and abnormal remodeling of extracellular matrix. This provides a new drug use option for aortic aging without relying on surgery or interventional treatment. By activating specific transcriptional regulatory pathways, comprehensive improvement of vascular wall structure and function can be achieved, which has the technical advantages of clear mechanism of action and clear target. This provides a new technological pathway for drug development and clinical translation of age-related aortic diseases and their complications.

[0024] In summary, this invention, through a novel application of the known compound Juglanin, solves the problem of the lack of targeted molecular intervention methods for aortic aging in the prior art, and has clear technical effects and application value. Attached Figure Description

[0025] Figure 1 The experimental design of the accelerated aortic aging model is shown in the diagram: KLF2 flox / flox control mice and KLF2SMKO mice were continuously infused with AngII (400 ng / kg / min) combined with Bleo (40 ng / kg / min) via ALZET® micro osmotic pump after surgery to induce accelerated aortic aging.

[0026] Figure 2 The results of PWV analysis are used to assess vascular stiffness and aging.

[0027] Figure 3 The results of β-galactosidase staining associated with aging of the entire aorta are used to detect the aging level of VSMCs.

[0028] Figure 4 Masson's trichrome staining reveals the collagen deposition in the aorta (blue), reflecting the degree of fibrosis.

[0029] Figure 5 Elastin-Vangisen (EVG) staining reveals elastic fibers (black) and ECM tissue, and provides corresponding quantitative analysis to assess vascular structural integrity.

[0030] Figure 6 The results of Western blot analysis of the expression levels of p16, FN1, α-SMA and SM22α proteins in the aorta.

[0031] Figure 7 For the results of quantitative analysis of protein expression (n=4), α-Tubulin was used as an internal control. Data are expressed as mean ± standard error (SEM).

[0032] Figure 8 Juglanin upregulated KLF2 mRNA expression.

[0033] Figure 9 The results of PWV analysis are used to assess vascular stiffness and aging.

[0034] Figure 10 The results of β-galactosidase staining associated with aging of the entire aorta are used to detect the aging level of VSMCs.

[0035] Figure 11 Masson's trichrome staining reveals the collagen deposition in the aorta (blue), reflecting the degree of fibrosis.

[0036] Figure 12 Elastin-Vangisen (EVG) staining reveals elastic fibers (black) and ECM tissue, and provides corresponding quantitative analysis to assess vascular structural integrity.

[0037] Figure 13 The results of Western blot analysis of the expression levels of p16, FN1, α-SMA and SM22α proteins in the aorta were obtained.

[0038] Figure 14 For the results of quantitative analysis of protein expression (n=4), α-Tubulin was used as an internal control. Data are expressed as mean ± standard error (SEM).

[0039] Figure 15 This is a statistical graph showing the changes in aortic diameter in the model.

[0040] Figure 16 This is a diagram showing the dissection results of the model. Detailed Implementation

[0041] Example 1: Study on the role of KLF2 in the in vivo aortic aging process To systematically investigate the role of the transcription factor KLF2 in VSMCs during aortic aging in vivo, a VSMC-specific KLF2-deficient mouse model (KLF2) was constructed. SMKO ): KLF2 flox / flox Mice and ACTA2-Cre ERT2 Transgenic mice were hybridized to obtain KLF2. flox / flox ACTA2-Cre ERT2 Offspring. Cre recombination was induced by daily intraperitoneal injection of tamoxifen for 7 consecutive days, successfully knocking out the KLF2 gene in VSMCs.

[0042] Eight weeks after gene knockout (mice were 9 weeks old), we treated mice with AngII in combination with Bleo to simulate an in vivo vascular aging environment. In the experiment, ALZET was used... ® A micro-osmotic pump continuously infused AngII (400 ng / kg / min) and Bleo (40 ng / kg / min) for 14 days to establish an aortic aging model. Figure 1To further assess changes in vascular function, we measured pulse wave velocity (PWV), the gold standard for arteriosclerosis and vascular aging. Results showed that AngII+Bleo treatment significantly increased PWV, while VSMC-specific KLF2 deficiency further exacerbated the increase in PWV. Figure 2 The results suggest that KLF2 plays a protective role in maintaining aortic elasticity and slowing vascular sclerosis. This finding is highly consistent with the clinically observed age-related decline in vascular compliance. At the cellular level, we assessed the aging status of vascular cells using aging-associated β-galactosidase (SA-β-gal) staining. The results showed that AngII+Bleo treatment led to a significant aging phenotype in VSMCs in the aorta, and KLF2 deficiency further amplified this effect, with a significant increase in aging-positive cells (…). Figure 3 Histological analysis further revealed the impact of KLF2 deficiency on vascular structure: Masson trichrome staining showed that AngII+Bleo treatment significantly increased collagen deposition in the vessel wall, suggesting enhanced vascular fibrosis; KLF2 SMKO Mice exhibited more significant collagen deposition, indicating that KLF2 deficiency exacerbated the profibrotic phenotype of blood vessels. Figure 4 Furthermore, the integrity of aortic elastic fibers was assessed by elastin-Vangisen (EVG) staining. The results showed that AngII+Bleo treatment induced elastin breakage and elastic layer destruction in the control group, while KLF2-deficient mice exhibited more severe elastic fiber damage, indicating that KLF2 plays a protective role in maintaining vascular structural integrity. Figure 5 Furthermore, we assessed the expression of key aging and phenotype-related proteins in the aorta. Western blot analysis showed that, compared with KLF2... flox / flox Compared with the control group, KLF2 SMKO The expression of the aging marker p16 was significantly increased in the mouse aorta, while the expression of the contractile VSMC markers α-SMA and SM22α was significantly decreased. Simultaneously, the expression level of the fibrosis marker FN1 was significantly increased. Figure 6 , Figure 7These results indicate that KLF2 deficiency not only accelerates the aging process of VSMCs but also promotes the phenotypic transformation from contractile to profibrotic VSMCs. In summary, VSMC-specific KLF2 deficiency significantly exacerbates AngII+Bleo-induced aortic aging in mice, including impaired vascular function (elevated PWV), enhanced cellular senescence (increased SA-β-gal positive cells), structural changes (increased collagen deposition and elastic fiber damage), and molecular phenotypic alterations (upregulation of p16, downregulation of α-SMA / SM22α, and upregulation of FN1). These results strongly suggest that KLF2 plays a crucial protective role in the aorta in vivo, and its deficiency amplifies the pathological features of vascular aging, providing important evidence for elucidating the mechanisms of vascular aging and potential intervention targets.

[0043] Example 2: Effects of the KLF2 agonist Juglanin on an animal model of aortic aging-related aortic aneurysm. Juglanin is a natural flavonoid derived from plants in the genus Juglans and has been reported to possess anti-inflammatory, antioxidant, and cellular aging-related signaling pathway regulatory activities. Juglanin treatment significantly upregulated the expression level of KLF2 in aortic tissue, suggesting that it has a direct or indirect activating effect on the KLF2 signaling pathway. Figure 8 In this study, juglanin was used to activate the KLF2 signaling pathway to evaluate its intervention effect on age-related structural and functional degeneration of the aorta. In the experimental design, 8-week-old mice received continuous infusion of AngII combined with Bleo to induce age-like changes in the aorta, and were treated with juglanin daily (30 mg / kg / day) to systematically evaluate the regulatory role of juglanin in the aortic aging process.

[0044] At the vascular function level, compared with the Vehicle group, Juglanin treatment significantly reduced AngII+Bleo-induced aortic pulse wave velocity (PWV) in mice. Figure 9 This indicates a significant improvement in aortic stiffness and restoration of vascular compliance. Overall morphological observation of the isolated aorta showed that Juglanin treatment significantly reduced aortic dilation and resulted in a more regular vascular appearance. Figure 10 This indicates that Juglanin intervention can effectively alleviate age-related vascular structural degeneration.

[0045] Histological analysis further confirmed the protective effect of juglanin against the aging phenotype of the aorta. Masson trichrome staining results showed that juglanin significantly reduced the proportion of collagen deposition in the vessel wall ( Figure 11The results suggest that age-related fibrosis was inhibited. EVG staining showed that elastic fibers in the aorta of Vehicle group mice were significantly broken, disordered, and degraded, while Juglanin treatment significantly improved the integrity of the elastic layer structure and significantly reduced the degree of elastic fiber degradation. Figure 12 This suggests that the mechanical support structure of the blood vessel wall is maintained.

[0046] At the molecular level, Western blot analysis showed that juglanin significantly inhibited aortic aging-related cellular phenotypic remodeling. Compared with the vehicle group, the expression of aging markers p16 and p21 was significantly decreased after juglanin treatment; simultaneously, the expression of vascular smooth muscle cell contractile phenotypic markers α-SMA and SM22α was significantly restored, while the expression of the pro-fibrosis-related extracellular matrix protein FN1 was significantly reduced. Figure 13 , Figure 14 The above results suggest that KLF2 signaling pathway imbalance is one of the important mechanisms driving VSMC age-related reprogramming, and that Juglanin can effectively reverse this pathological process by activating KLF2 signaling.

[0047] Meanwhile, this invention establishes an animal model of senescence-associated abdominal aortic aneurysm (saAAA) and uses it to evaluate the intervention effect of juglanin on aortic dilation and aneurysm progression.

[0048] Specifically, aged mice (typically 18–22 months old) were used as the baseline aging background. In addition, continuous infusion of angiotensin II (AngII) and treatment with β-aminopropionitrile (BAPN) were administered to induce further damage to the elastic structure of the aortic wall and impaired extracellular matrix cross-linking in aging vessels, thus constructing an aortic aneurysm model with a clearly defined aging background. This model more significantly simulated the pathological features of age-related increased vascular wall fragility, elastic fiber rupture, and accelerated aortic dilation compared to the traditional AngII model.

[0049] After the model was established, mice were randomly divided into a control group (Vehicle group) and a Juglanin intervention group. Results showed that in the aging-related AngII+BAPN-induced aortic aneurysm model, the aorta of mice in the Vehicle group was significantly dilated, characterized by a significant increase in vessel diameter and a marked enlargement of the aneurysm structure; while after Juglanin treatment, the degree of aortic dilation was significantly reduced, and the maximum aortic diameter was significantly decreased, suggesting that Juglanin can effectively inhibit the progression of aortic aneurysms in the context of aging. Figure 15 Ultrasound examination further confirmed that, compared with the Vehicle group, the aortic diameter in the Juglanin group was significantly reduced (P = 0.013), suggesting that it has a clear inhibitory effect on hemodynamically related aortic dilatation. Simultaneously, gross anatomical results showed that Juglanin treatment significantly improved aortic tortuosity and focal aneurysmal dilatation, and reduced the degree of aneurysm formation. Figure 16 The "aged mouse + AngII + BAPN" model established in this invention can effectively simulate the development and progression of age-related aortic aneurysms, and Juglanin can significantly inhibit aortic dilation and aneurysm formation in this model, suggesting its potential application value in the prevention and treatment of age-related vascular degenerative diseases.

[0050] In summary, this study demonstrates that the KLF2 signaling pathway plays a crucial regulatory role in aortic aging. Targeted activation via Juglanin effectively improves functional aortic stiffness, reduces fibrosis and elastic laminar degeneration, and inhibits VSMC age-related reprogramming, thereby delaying the overall aging process of the aorta. These results provide new theoretical basis and potential treatment strategies for pharmacological intervention in age-related aortic diseases.

Claims

1. Application of KLF2 agonists in the preparation of drugs to alleviate aortic aging.

2. In the application according to claim 1, the drug provides one or more of the following effects: (1) Inhibit age-related molecular phenotypic abnormalities and functional imbalances in vascular smooth muscle cells; (2) Regulate the abnormal secretion and deposition of extracellular matrix, and reduce pathological remodeling of the aortic wall; (3) Promote the expression of genes related to vascular smooth muscle cell contraction and maintain or restore the integrity of the aortic media structure.

3. In the application according to claim 2, the aging-related molecular phenotypic abnormality is upregulation of p16 and / or p21 expression; and / or regulation of abnormal secretion and deposition of extracellular matrix includes reduced FN1 expression; and / or the vascular smooth muscle cell contraction-related gene is α-SMA and / or SM22α.

4. Application of KLF2 agonists in the preparation of drugs for the treatment or prevention of aortic dilatation.

5. Application of KLF2 agonists in the preparation of drugs for the treatment or prevention of aortic aging-related aneurysms.

6. The application according to any one of claims 1-5, wherein the KLF2 agonist is Juglanin.

7. In the application according to claim 6, Juglanin is the sole active ingredient in the drug.

8. The application according to claim 6, wherein the drug is an oral or injectable dosage form.

9. The application according to claim 8, wherein the drug is an oral dosage form.

10. The application according to any one of claims 1-5, wherein the medicament further comprises a pharmaceutically acceptable excipient.

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