Use of chlorogenic acid in preparation of drugs for preventing or treating aortic dissection

Abstract

The application relates to the field of medicine, in particular to application of chlorogenic acid or a pharmaceutically acceptable salt thereof in preventing, inhibiting or treating aortic dissection. The application first discovers that chlorogenic acid has a remarkable effect in treating aortic dissection, and provides a brand-new thought and method for treating aortic dissection. In addition, compared with traditional surgical operation and interventional treatment, the treatment cost is relatively low by using chlorogenic acid, the economic burden of patients is reduced, and the consumption of medical resources is also reduced.

Description

Technical Field

[0001] This invention relates to the field of medicine, specifically to the application of chlorogenic acid in the prevention or treatment of aortic dissection. Background Technology

[0002] Aortic dissection (AD), also known as aortic aneurysm dissection, is a serious cardiovascular emergency. It occurs when a tear appears in the intima of an aorta, allowing blood to enter the arterial wall and form a hematoma. This hematoma can further dissect the intima and media of the aorta, resulting in aortic dissection. The generally accepted causes are structural abnormalities of the aortic media itself and hemodynamic abnormalities. High-risk groups include those with hypertension, older age, aortic atherosclerosis, and hereditary vascular diseases. The disease progresses rapidly, has a high early mortality rate, and its incidence is related to population aging.

[0003] Currently, treatment methods for aortic dissection mainly include drug therapy, surgery, and interventional therapy. However, existing treatments have certain limitations. Surgery is high-risk and invasive, and has stringent requirements on the patient's physical condition; while interventional therapy is less invasive, its applicability is limited, and it requires advanced technical skills.

[0004] In terms of drug treatment, commonly used drugs are mainly used to control blood pressure and heart rate to reduce the risk of aortic dissection rupture, but their effect on repairing the lesions of aortic dissection itself is limited.

[0005] Therefore, there is an urgent need to provide an effective, safe, and easy-to-use treatment option for aortic dissection. Summary of the Invention

[0006] (a) Technical problems to be solved

[0007] To address the shortcomings of existing technologies, this invention provides an application of chlorogenic acid in the prevention and treatment of aortic dissection, solving the technical problem that the number of drugs currently available for the prevention and treatment of aortic dissection is very limited.

[0008] (II) Technical Solution

[0009] To achieve the above objectives, the present invention provides the following technical solution:

[0010] On the one hand, the present invention provides the use of chlorogenic acid (CGA) or a pharmaceutically acceptable salt thereof in the preparation of drugs for the prevention, inhibition or treatment of aortic dissection.

[0011] In one embodiment, the pharmaceutically acceptable salt of chlorogenic acid includes sodium chlorogenicate, potassium chlorogenicate, or calcium chlorogenicate.

[0012] In one aspect, the present invention provides the use of chlorogenic acid or a pharmaceutically acceptable salt thereof as the main active ingredient in the preparation of a medicament for the prevention, inhibition or treatment of aortic dissection.

[0013] In a second aspect, the present invention provides a pharmaceutical composition for treating aortic dissection, the pharmaceutical composition comprising a therapeutically effective amount of chlorogenic acid or a pharmaceutically acceptable salt thereof.

[0014] In one embodiment, the method includes the combined use of chlorogenic acid or a pharmaceutically acceptable salt thereof with other drugs for treating aortic dissection, wherein the other drugs for treating aortic dissection are selected from at least one of antihypertensive drugs and beta-blockers.

[0015] In one embodiment, the medicament for treating aortic dissection further includes pharmaceutically acceptable excipients.

[0016] In one aspect, the present invention provides a pharmaceutical preparation for treating aortic dissection, the pharmaceutical preparation comprising the above-described pharmaceutical composition.

[0017] In one embodiment, the pharmaceutical formulation includes a solid dosage form, a semi-solid dosage form, a liquid dosage form, or a gaseous dosage form.

[0018] In one embodiment, the solid dosage form includes powders, pills, tablets, and capsules; the semi-solid dosage form includes ointments, suppositories, and gels; the liquid dosage form includes lotions, mixtures, solutions, and injections; and the gaseous dosage form includes aerosols and sprays.

[0019] In one aspect, the present invention provides the use of the above-described pharmaceutical composition or pharmaceutical preparation in the preparation of a medicament for the prevention and / or treatment of aortic dissection.

[0020] In one embodiment, the above-mentioned chlorogenic acid or its pharmaceutically acceptable salt is used as the main active ingredient in a pharmaceutical composition or pharmaceutical preparation.

[0021] In one embodiment, chlorogenic acid or a pharmaceutically acceptable salt thereof as the main active ingredient means that chlorogenic acid or a pharmaceutically acceptable salt thereof accounts for more than 10% of the active ingredient; preferably, more than 20%; preferably, more than 30%; preferably, more than 40%; preferably, more than 50%; preferably, more than 60%; preferably, more than 65%; preferably, more than 70%; preferably, more than 75%; preferably, more than 80%; preferably, more than 85%; preferably, more than 90%; preferably, more than 95%.

[0022] In one aspect, the present invention provides a method for treating a patient with aortic dissection, comprising administering to the patient a therapeutically effective amount of chlorogenic acid or a pharmaceutically acceptable salt thereof.

[0023] (III) Beneficial Effects

[0024] This invention provides the application of chlorogenic acid in the prevention and treatment of aortic dissection. Compared with the prior art, it has the following beneficial effects:

[0025] This invention is the first to discover the significant effect of chlorogenic acid in treating aortic dissection, providing a novel approach and method for the treatment of aortic dissection. Furthermore, compared to traditional surgical and interventional treatments, the use of chlorogenic acid is relatively low-cost, reducing the financial burden on patients and also decreasing the consumption of medical resources. Attached Figure Description

[0026] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0027] Figure 1 : Figure 1 a. Experimental design. Four-week-old male C57BL / 6J mice were treated with water or 0.4% BAPN and infused with physiological saline or angiotensin II (Ang). II; 1000 ng / kg / min), monitor aortic dilation and blood pressure, and euthanize at designated time points; 1b, representative aortic photographs of each group at the end of the experiment and the incidence of aortic dissection (AD) in each group, CON (n=10), AD (n=12), CGA (n=10), Fisher's exact test; 1c, survival rate of mice in each group; 1d, representative ultrasound images and quantification of the maximum aortic diameter in each group; n=5; 1e, body weight of mice during the experiment, CON (n=10), AD (n=12 on day 20, n=11 on day 23, n=10 before day 27, n=9 on day 28, n=3 on day 29), CGA (n=10 on day 28, n=9 on day 29); 1f, diastolic blood pressure of mice during the experiment; n=5. *p<0.05, **p<0.01, ***p<0.001.

[0028] Figure 2 : Figure 2 a) Representative H&E, Masson (collagen content), and EVG (elastin breakage grade) staining of aortic sections; n=5; 2b) Representative immunofluorescence staining and quantification of α-SMA (red), cell nuclei stained with DAPI (blue); n=6. *p<0.05, **p<0.01, ***p<0.001.

[0029] Figure 3 : Figure 3 a. Volcano plot comparing protein expression changes between CON (control group) and AD (aortic dissection group) (p<0.05); 3b. Volcano plot comparing protein expression changes between AD (aortic dissection group) and CGA (Cardiovascular Aortic Dissection Group) (p<0.05); 3c. Venn diagram illustrating the overlap between upregulated proteins in AD and downregulated proteins in CGA; 3d. Heatmap of different protein expression levels, with low expression shown in blue and high expression shown in red; 3e. Unbiased clustering of 79,544 cells from all nine samples revealed eight cell clusters; 3f. SLC43A1 expression in smooth muscle cells (SMCs) of the AD group was significantly higher than that of the CON group; 3g. Immunofluorescence staining of LAT3 (green) and α-SMA (red) (bottom), with cell nuclei stained with DAPI (blue). *p<0.05, **p<0.01, ***p<0.001.

[0030] Figure 4 : Figure 4 a) qPCR analysis of ACTA2 and OPP1 in human aortic smooth muscle cells (HASMCs) treated with PDGF-BB (0, 20, 30, 40, 50, 60 ng / mL, for 24 hours); n=4; 4b) qPCR analysis of SLC43A1 in HASMCs treated with PDGF-BB (0, 20, 30, 40 ng / mL, for 24 hours); n=4; 4c) qPCR analysis of SLC43A1 in HASMCs treated with PDGF-BB (40 ng / mL, for 24 hours) and CGA (0, 10, 20, 40, 8... qPCR analysis of ACTA2 and OPP1 was performed on HASMCs treated with 0 mm (treated for 24 hours); n=4; 4d, qPCR analysis of SLC43A1 was performed on HASMCs treated with PDGF-BB (40 ng / mL, treated for 24 hours) and CGA (0, 10, 20, 40, 80 mm, treated for 24 hours); n=4; 4e, representative images and quantitative analysis of wound healing assay; n=3, scratch wound assay was performed on three groups of HASMCs, and images were taken at 0 and 24 hours after scratching (white lines indicate wound edges). The migration area of ​​HASMCs was analyzed from the average microscopic field of view. *p<0.05, **p<0.01, ***p<0.001.

[0031] Figure 5 : Figure 5a-5d represent the branched-chain amino acid (BCAA) levels in aortic tissue, serum samples, human aortic smooth muscle cells (HASMCs), and culture medium, respectively; n=6; 5e,f, expression of slc43a1 and α-SMAm RNA was determined by qRT-PCR; n=4. 5g-5h, Western blot and quantitative density analysis of LAT3, α-SMA, and phosphorylated mTOR; n=6; 5i, representative immunofluorescence staining of p-mTOR (green) and α-SMA (red) in each aortic group; cell nuclei were stained with DAPI (blue). *p<0.05, **p<0.01, ***p<0.001.

[0032] Figure 6 : Figure 6 a) AR mRNA expression was determined by qRT-PCR; 6b) Immunohistochemical staining of AR. Scale bar = 20 mm; 6c) Potential binding sites of AR on the SLC43A1 promoter were predicted using JASPER and HumanTFDB, along with potential binding sites and corresponding mutations of AR to the SLC43A1 promoter sequence; 6d, e) Luciferase activity was detected using a dual-luciferase assay; n = 6. *p<0.05, **p<0.01, ***p<0.001.

[0033] Figure 7 : Figure 7 a, 7b, Immunofluorescence staining of BCKDK / pBCKDHA (green) and α-SMA (red) (bottom), cell nuclei stained with DAPI (blue); 7c, qRT-PCR determination of BCKDK mRNA expression, n=4; 7d, Western blot and quantitative density analysis of BCKDK and phosphorylated BCKDHA; n=6. *p<0.05, **p<0.01, ***p<0.001. Detailed Implementation

[0034] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are described clearly and completely. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0035] This invention provides an application of chlorogenic acid in the prevention and treatment of aortic dissection, solving the technical problem that the current drugs available for the prevention and treatment of aortic dissection are very limited.

[0036] definition:

[0037] The term "suppression" refers to reducing a measurable amount or preventing it entirely.

[0038] The term "therapeuticly effective amount" refers to the amount of chlorogenic acid or a pharmaceutically acceptable salt thereof that is effective in treating a disease or condition in mammals. In the case of cancer, a therapeutically effective amount of chlorogenic acid or a salt thereof can reduce the number of cancer cells; reduce tumor size; inhibit (i.e., to some extent slow down and preferably stop) the invasion of cancer cells into surrounding organs; inhibit (i.e., to some extent slow down and preferably stop) tumor metastasis; inhibit tumor growth to some extent; and / or alleviate one or more symptoms associated with cancer to some extent. In terms of the extent to which the drug can inhibit growth and / or kill existing cancer cells, it can be cytoseptic and / or cytotoxic. For cancer treatment, efficacy can be measured, for example, by assessing time to progression (TTP) and / or determining the response rate (RR).

[0039] The term "patient" refers to a subject who has been given chlorogenic acid or a salt thereof according to the present invention. Patients include, but are not limited to, humans, rats, mice, guinea pigs, non-human primates, pigs, goats, cattle, horses, dogs, cats, birds, and poultry. Typically, patients are rats, mice, dogs, humans, or non-human primates, but more commonly, humans.

[0040] The term "treatment" refers to both therapeutic and preventative treatment aimed at suppressing or slowing (alleviating) undesirable physiological changes or conditions, such as the development or spread of cancer. For the purposes of this invention, beneficial or desired clinical outcomes include, but are not limited to, reduction of symptoms, lessening of disease severity, stabilization of the disease state (i.e., non-deterioration), delay or slowing of disease progression, improvement or mitigation of the disease state, and remission (whether partial or complete), whether detectable or undetectable. "Treatment" may also imply a prolonged survival compared to expected survival without treatment. Those requiring treatment include both those already suffering from the disease or condition and those susceptible to it.

[0041] Within the context of cancer, the term "treatment" includes any or all of the following: killing tumor cells; inhibiting the growth of tumor cells, cancer cells, or tumors; inhibiting the replication of tumor cells or cancer cells; reducing the overall tumor burden or the number of cancer cells; and improving one or more symptoms of accompanying diseases.

[0042] The phrase “its salt” as used in this article refers to the salt form of the compound.

[0043] As used herein, "chlorogenic acid or a pharmaceutically acceptable salt thereof as the main active ingredient" means that chlorogenic acid or a pharmaceutically acceptable salt thereof accounts for more than 10% of the active ingredient; preferably, more than 20%; preferably, more than 30%; preferably, more than 40%; preferably, more than 50%; preferably, more than 60%; preferably, more than 65%; preferably, more than 70%; preferably, more than 75%; preferably, more than 80%; preferably, more than 85%; preferably, more than 90%; preferably, more than 95%.

[0044] The following detailed description uses specific examples.

[0045] The chlorogenic acid (CGA) [drug catalog number: Cat.#HY-N0055] involved in the examples was purchased from MedChemexpres; the C57BL / 6 male mice (4 weeks old) were all purchased from Henan Scobes Biotechnology Co., Ltd.; all experiments were conducted at the SPF-grade animal experimental center of the First Affiliated Hospital of the University of Science and Technology of China, with the ambient temperature controlled at 22-24℃, and the experimental mice allowed free access to food and water; all animal experiments complied with the regulations of the Animal Ethics Committee of the First Affiliated Hospital of the University of Science and Technology of China.

[0046] Data are expressed as mean ± standard error (M ± SEM). Statistical analysis was performed using GraphpadPrism 8 software. One-way ANOVA was used for comparisons between two groups, and one-way ANOVA was also used for comparisons between more than two groups. Newman-Coles multiple comparison tests were then performed. A p-value < 0.05 was considered statistically significant.

[0047] Example 1: Establishment of an AD animal model

[0048] Thirty-two four-week-old C57 mice were randomly divided into three groups using statistical methods: a CON control group, an AD model group, and a CGA treatment group. Mice in the CON group were given double-distilled water for 28 days, and on day 28, a micro-osmotic pump (Alzet, model 1003D) containing physiological saline was implanted subcutaneously. Mice in the AD group were given double-distilled water containing 0.4% BAPN (Sigma-Aldrich, A3134, JAPAN) for 28 days, followed by an infusion of 1000 ng / kg / min angiotensin II (MCE, Cat, #HY-13948) on day 28 via the micro-osmotic pump. Mice in the CGA group, in addition to the AD model, received daily intraperitoneal injections of CGA 50 mg / kg. During the experiment, the time and cause of death for all mice were recorded. After the experiment, all mice were euthanized, and the aorta was dissected; the aortic lesions were recorded under a stereomicroscope.

[0049] like Figure 1a, b, c: After CGA treatment, at 29 days, the incidence of AD induced by BAPN and Ang II significantly decreased from 83% (10 / 12) to 40% (4 / 10), and the survival rate significantly increased from 25% (3 / 12) to 70% (7 / 10).

[0050] Example 2: Routine echocardiography and blood pressure measurement

[0051] Transthoracic echocardiography was performed using a Visual Sonics Vevo 2100 system equipped with an MS400 probe. Aortic arch width was measured at the aortic level in mice. Anesthesia was induced by 3% isoflurane and confirmed by no response to stimulation of one of the hind paws. During echocardiographic acquisition, isoflurane was reduced to 1.0–1.5% under controlled body temperature conditions and adjusted to maintain a heart rate of 415–460 beats per minute. All mice recovered successfully after the examination.

[0052] like Figure 1 d: CGA treatment reduced aortic widening induced by BAPN and Ang II. e.g., 1f: CGA treatment improved aortic wall stiffness induced by BAPN and Ang II.

[0053] Example 3: Histochemical Analysis

[0054] Mouse aortic tissue was fixed in 4% paraformaldehyde for at least 24 hours, dehydrated in 30% sucrose, embedded in OCT, and then prepared into 5μm frozen sections. H&E, Masson, and EVG staining were performed.

[0055] like Figure 2 a: HE staining showed erythrocytes penetrating the lumen and filling the separated vascular layer. Masson staining showed disordered collagen fiber tissue in the AD group. Electron microscopy staining showed significant disorder and breakage of elastic structures. The vascular structure in the CON group was intact. Although the aortic wall in the CGA group was thicker than that in the CON group, no obvious false lumen formation was observed, and no obvious erythrocyte infiltration was observed within the wall. The disordered collagen arrangement and broken elastin fibers were significantly reduced compared to the AD group.

[0056] Example 4: Immunohistochemical detection of expression levels of HASMC phenotypic switching-related proteins

[0057] Mouse aortic tissue was fixed in 4% paraformaldehyde for at least 24 hours, dehydrated with 30% sucrose, embedded in OCT, and then prepared into 5μm frozen sections. Immunofluorescence staining was performed to detect the expression of α-SMA and p-mTOR.

[0058] like Figure 2 b: Arterial cross-section of AD group αα-SMA expression was significantly lower in the CGA group than in the CON group, while α-SMA expression in the CGA group essentially returned to normal levels. In the AD group, upregulated p-mTOR co-localized with the cell nucleus, and CGA treatment reversed this phenomenon. These results indicate that CGA treatment can improve AD-induced aortic smooth muscle phenotypic transformation and mTOR pathway activation.

[0059] Example 5: Exploration of potential targets for chlorogenic acid in the treatment of AD

[0060] Unbiased proteomics screening was performed on aortic samples from three mice in the CON, AD, and CGA groups. Overall, comparing the data from the CON and AD groups, 44 proteins showed differential expression (p<0.05, Log2 (folding change) >2 or <-2), including 29 upregulated proteins and 15 downregulated proteins. Figure 3 a). Meanwhile, data comparison between the AD group and the CGA group showed that 25 proteins were differentially expressed (p<0.05, Log2(fractal)>2 or<-2), including 1 upregulated protein and 24 downregulated proteins. Figure 3 b). 23 overlapping proteins were found among the 29 upregulated proteins (CON vs AD) and 24 downregulated proteins (AD and CGA). Figure 3 cd). Simultaneously, this invention analyzed single-cell RNA sequencing data (GSE213740) from aortic samples collected from 6 AD patients and 3 non-AD control groups. Cells were classified into 8 major cell types ( Figure 3 e). This invention focuses particularly on differentially expressed genes in the aforementioned overlapping proteins. We randomly selected 4000 cells from the CON and AD groups respectively and found that SoluteCarrier Family 43 (Amino Acid System L Transporter) (slc43a1) was significantly upregulated in the AD group. Figure 3 f). slc43a1 encodes the small subunit 3 (LAT3) protein, a large neutral amino acid transporter located in the muscle plasma membrane, which mediates the transport of neutral amino acids such as L-leucine, L-isoleucine, and L-valine. To further verify the importance of LAT3, immunofluorescence staining was performed to determine LAT3 expression. The results of this invention clearly show that LAT3 is increased in the AD group, but reversed after CGA treatment. Figure 3 g).

[0061] Example 6: Chlorogenic acid inhibits PDGF-BB-induced HASMC phenotypic conversion. In order to study the role of LAT3 in CGA-mediated anti-AD protection, this invention uses (human arterial smooth muscle cells) HASMC for in vitro experiments. α-SMA is a marker for SMCs that maintain the contractile phenotype, while secretory phosphoprotein 1 (SPP1) is a marker for the differentiation phenotype. This invention used concentration gradients (20, 30, 40, 50, 60 ng / mL) to determine the effect of PDGF-BB, finding that treatment with 40 ng / mL PDGF-BB... α -SMA expression was the lowest, and SPP1 expression was the highest. Figure 4 a). Then, the effects of CGA were determined using concentration gradients (10, 20, 40, 80 μM) and co-treated with 40 ng / mL PDGF-BB. The present invention found that 40 μM CGA maximally reversed the effect of PDGF-BB on SMC phenotypic conversion. Figure 4 c). Simultaneously, CGA can reverse PDGF-BB-induced slc43a1 transcriptional increase in a concentration-dependent manner. Figure 4 (b, d). Furthermore, PDGF-BB treatment showed increased migration in HASMCs, and combined treatment with CGA restored HASMCs to a steady state. Figure 4 e). In summary, CGA reversed PDGF-BB-induced SMC phenotypic transformation and slc43a1 transcriptional increase.

[0062] High levels of BCAAs (including L-leucine, L-isoleucine, and L-valine) maintain sustained activation of mTOR signaling, a mechanism target of rapamycin kinase, which is associated with various diseases. Liming Yu et al. reported that, compared to controls, BCAA levels were significantly increased in the aorta of AD patients, in a BAPN-induced mouse aortic dissection model, and in a TNF-α-induced SMC reprogramming model. This invention hypothesizes that CGA can inhibit the upregulation of slc43a1 and the overexpression of LAT3 on the aortic SMC membrane, thereby further reducing the accumulation of excess branched-chain amino acids in SMCs and thus improving AD.

[0063] Example 7: CGA treats AD through the LAT3-mTOR pathway

[0064] The levels of BCAAs in the aorta and serum of mice in the CON, AD, and CGA groups were measured. The results showed that, compared with the CON group, the levels of BCAAs in the aortic tissue of mice in the AD group were significantly increased. Figure 5 a), while CGA treatment reduced the levels of BCAAs in mouse aortic tissue and serum ( Figure 5 a, b). Consistent with in vivo results, PDGF-BB treatment increased the content of BCAAs in SMCs, while CGA treatment reversed this result. Figure 5c). Meanwhile, the BCAA content in the cell culture medium of PDGF-BB treated wells was significantly lower than that of the CON group. The BCAA content in the cell culture medium of CGA treated wells recovered somewhat, but this was not statistically significant. Figure 5 d).

[0065] The above results indicate that the BCAA content in SMCs may be a key factor affecting AD. Then, this invention co-cultured cells with exogenous BCAA, PDGF-BB, and CGA. PDGF-BB stimulation increased LAT3 expression at both RNA and protein levels in SMCs, and CGA treatment reversed this result. However, incubating SMCs with BCAA concurrently with CGA treatment attenuated the therapeutic effect of CGA, although this attenuation was not statistically significant. Figure 5 e, g). Then, this invention observed the phosphorylation of mTOR and the expression of α-SMA in the CON, AD, CGA, and BCAA groups. Compared with the CGA group, the phosphorylation level of mTOR in the BCAA group was significantly upregulated, almost equivalent to that in the AD group ( Figure 5 (g) Consistent with this, PDGF-BB stimulation significantly reduced α-SMA expression, leading to cell dedifferentiation, while CGA treatment restored α-SMA expression. However, CGA treatment combined with BCAA restored cells to the dedifferentiation state. This implies that BCAA, together with CGA, confers and almost completely eliminates the protective effect of CGA on HASMCs. Figure 5 f, g).

[0066] To further investigate whether the protective effect induced by CGA is related to LAT3, this invention transfected HASMCs with the LAT3-OE plasmid to overexpress LAT3. The results showed that LAT3 overexpression significantly reversed the protective effect of CGA on HASMCs. On the basis of PDGF-BB stimulation, LAT3 overexpression and mTOR phosphorylation further increased, while the expression of contraction markers in HASMCs was statistically significantly reduced. Figure 5 h). Consistent with HASMC data, in the AD group, upregulated pmTOR co-localized with the cell nucleus, while CGA treatment reversed this upregulation (h). Figure 5 i). In summary, these results indicate that CGA protects mice from AD through the LAT3-mTOR pathway.

[0067] Example 8: CGA inhibits the transcription of slc43a1 by suppressing the expression of transcription factor AR.

[0068] Previous studies have reported that the androgen receptor (AR) is a transcription factor that controls the transcription and expression of slc43a1. This invention preliminarily found that CGA inhibits the upregulation of AR transcription levels induced by PDGF-BB stimulation of HASMCs. Figure 6 a) Immunohistochemical results showed that, compared with the AD group, the expression of AR in the cell nuclei of the CGA group was reduced ( Figure 6 b). It is speculated that the inhibitory effect of CGA on slc43a1 depends on the disappearance of AR.

[0069] This invention uses JASPER and HumanTFDB to predict the binding site of AR on the slc43a1 promoter. Figure 6 c). AR-OE was simultaneously constructed into the pcDNA3.1 vector. To determine promoter activity, HEK 293T cells (approximately 60-70% confluence) were transfected with DNA containing slc43a1, AR-OE, and Renilla (for internal normalization) using Lipofectamine 8000™ Transfection Reagent (China). Luciferase activity was significantly increased in both the slc43a1-WT+pcDNA3.1 and slc43a1-WT+AR groups compared to the pGL3-Basic+pcDNA3.1 group. Luciferase activity was further increased in the slc43a1-WT+AR group compared to the slc43a1-WT+pcDNA3.1 group. Figure 6 d). These data indicate that AR can directly bind to the slc43a1 promoter and participate in slc43a1 transcription. Furthermore, this invention also mutated the predicted binding site of AR on the human slc43a1 promoter. Results showed that luciferase activity was reduced in the slc43a1-MT+pcDNA3.1 group compared to the slc43a1-WT+pcDNA3.1 group; similarly, luciferase activity was also reduced in the slc43a1-MT+AR group compared to the slc43a1-WT+AR group. These results indicate that AR can bind to the TFBS of the slc43a1 promoter and regulate slc43a1 transcription.

[0070] To further elucidate the molecular mechanism by which CGA inhibits LAT3 expression, this invention transfected slc43a1-WT or slc43a1-MT into HASMCs and examined the promoter activity after CGA treatment. Mutations at the slc43a1-AR binding site weakened the basal level of slc43a1 promoter activity, and also weakened the inhibitory effect of CGA on slc43a1 promoter activity. Figure 6 e). The results showed that CGA inhibits slc43a1 expression at the transcriptional level by activating the AR pathway.

[0071] Example 9: CGA may improve BCAA metabolism in HASMCs

[0072] Previous studies have reported that AD is accompanied by BCAA metabolic disorder in SMCs, and the expression of branched-chain ketoacid dehydrogenase kinase (BCKDK) and phosphorylation of branched-chain ketoacid dehydrogenase E1,α-peptide (BCKDHA) are important rate-limiting enzymes in BCAA metabolism. Therefore, this invention further investigates the effect of CGA on BCKDK and BCKDHA expression. First, immunofluorescence staining was performed on vascular sections from the CON group, AD group, and CGA group. It was found that compared with the CON group, the expression of BCKDK and phosphorylation of BCKDHA were significantly upregulated in the AD group, while CGA reversed this upregulation. Figure 7 (a, b). To investigate the changes in the expression of these two proteins, SMCs were treated using the methods described in the experiments above. Consistent with previous studies, PDGF-BB stimulation increased BDKDK expression and BCKDHA phosphorylation, indicating impaired branched-chain amino acid metabolism, which was reversed by functional incubation with CGA. Surprisingly, co-incubation of BCAA with PDGF-BB and CGA only alleviated CGA-induced BCKDK downregulation at the gene level, without mitigating the inhibitory effect of CGA on PDGF-BB damage; instead, it further reduced BCKDK expression and BCKDHA phosphorylation. Subsequently, it was found that PDGF-BB stimulation led to increased BCKDK expression and BCKDHA phosphorylation in SMCs, while CGA treatment reversed both increases. However, LAT3 overexpression had no effect on the changes in BCKDK and pBCKDHA. Figure 7 (c, d). These results suggest that CGA treatment may affect BCKDK and the LAT3-mTOR pathway, but there is no link between LAT3 and BCKDK.

[0073] In summary, this invention is the first to discover the significant effect of chlorogenic acid in treating aortic dissection, providing a novel approach and method for the treatment of aortic dissection. Furthermore, compared with traditional surgical and interventional treatments, the use of chlorogenic acid for treatment is relatively low-cost, reducing the economic burden on patients and also decreasing the consumption of medical resources.

[0074] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitations, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.

[0075] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. The use of a pharmaceutical composition in the preparation of a drug for the prevention, inhibition, or treatment of aortic dissection, characterized in that, The pharmaceutical composition comprises the combined use of chlorogenic acid or a pharmaceutically acceptable salt thereof with other drugs for treating aortic dissection; the other drugs for treating aortic dissection are selected from at least one of antihypertensive drugs and beta-blockers.

2. The application as described in claim 1, characterized in that, The pharmaceutical composition for treating aortic dissection also includes pharmaceutically acceptable excipients.

3. The use of a pharmaceutical formulation in the preparation of drugs for the prevention, inhibition, or treatment of aortic dissection, characterized in that, The pharmaceutical preparation includes the pharmaceutical composition according to claim 1 or 2.

4. The application as described in claim 3, characterized in that, The pharmaceutical preparations include solid dosage forms, semi-solid dosage forms, liquid dosage forms, or gaseous dosage forms.

5. The application as described in claim 4, characterized in that, The solid dosage forms include powders, pills, tablets, and capsules; the semi-solid dosage forms include ointments, suppositories, and gels; the liquid dosage forms include solutions and injections; and the gaseous dosage forms include aerosols and sprays.

6. The application as described in claim 4, characterized in that, The drug formulation type is tablet, capsule, or injection.

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