Application of chlorogenic acid in preparation of medicine for preventing or treating aortic dissection

Abstract

The invention relates to the field of medicine, in particular to application of chlorogenic acid or pharmaceutically acceptable salt thereof in preventing, inhibiting or treating aortic dissection. The obvious effect of chlorogenic acid in treating the aortic dissection is found for the first time, and a brand new thought and method are provided for treating the aortic dissection. Besides, compared with the traditional surgical operation and interventional therapy, the chlorogenic acid is relatively low in treatment cost, so that the economic burden of a patient is relieved, and meanwhile, the consumption of medical resources is also reduced.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and in particular to the application of chlorogenic acid in preventing or treating aortic dissection. Background Art

[0002] Aortic dissection (AD), also known as aortic dissecting aneurysm, is a serious cardiovascular emergency. When a rupture occurs in the intima of the arterial wall, blood enters the arterial wall through the rupture to form a hematoma, and further peels off the intima and media of the aorta, aortic dissection will occur. The generally recognized cause of the disease is the structural abnormality and hemodynamic abnormality of the aortic media itself, and the high-risk groups include patients with hypertension, older age, aortic atherosclerosis and hereditary vascular diseases. The disease progresses rapidly, with a high early mortality rate, and the incidence rate is related to the increase in population age.

[0003] At present, the treatment methods for aortic dissection mainly include drug therapy, surgery and interventional therapy. However, the existing treatment methods have certain limitations. Surgery is high-risk and traumatic, and requires strict physical conditions of patients; although interventional therapy is less invasive, it has a limited scope of application and high technical requirements.

[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 the effect on repairing 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] 1. Technical issues to be resolved

[0007] In view of the deficiencies of the prior art, the present invention provides an application of chlorogenic acid in the prevention and treatment of aortic dissection, which solves the technical problem that the current drugs for the prevention and treatment of aortic dissection are very limited.

[0008] (II) Technical solution

[0009] To achieve the above objectives, the present invention is implemented through the following technical solutions:

[0010] In one aspect, the present invention provides a use of chlorogenic acid (CGA) or a pharmaceutically acceptable salt thereof in the preparation of a drug for preventing, inhibiting or treating aortic dissection.

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

[0012] In one aspect, the present invention provides a use of chlorogenic acid or a pharmaceutically acceptable salt thereof as a main active ingredient in the preparation of a drug for preventing, inhibiting or treating aortic dissection.

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

[0014] In one embodiment, chlorogenic acid or a pharmaceutically acceptable salt thereof is used in combination 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 drug for treating aortic dissection further includes a pharmaceutically acceptable excipient.

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

[0017] In one embodiment, the pharmaceutical formulation comprises a solid dosage form, a semisolid dosage form, a liquid dosage form, or a gaseous dosage form.

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

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

[0020] In one embodiment, the chlorogenic acid or a pharmaceutically acceptable salt thereof is used as a 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 of 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] The present invention provides an application of chlorogenic acid in preventing and treating aortic dissection. Compared with the prior art, it has the following beneficial effects:

[0025] The present invention is the first to discover the significant effect of chlorogenic acid in treating aortic dissection, providing a new idea and method for the treatment of aortic dissection. In addition, compared with traditional surgical operations and interventional treatments, the cost of using chlorogenic acid for treatment is relatively low, which reduces the economic burden on patients and also reduces the consumption of medical resources. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[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 saline or angiotensin II (Ang II). II; 1000ng / kg / min), aortic dilatation and blood pressure were monitored, and euthanasia was performed at the indicated time points; 1b, representative photographs of the aorta in 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 fragmentation grade) staining of aortic sections; n = 5; 2b, Representative immunofluorescence staining and quantification of α-SMA (red), and nuclei were stained with DAPI (blue); n = 6. *p < 0.05, **p < 0.01, ***p < 0.001.

[0029] Figure 3 : Figure 3 a, Volcano plot of protein expression changes in the comparison between CON (control group) and AD (aortic dissection group) (p < 0.05); 3b, Volcano plot of protein expression changes in the comparison between AD (aortic dissection group) and CGA (p < 0.05); 3c, Venn diagram illustrating the overlap between AD upregulated proteins and CGA downregulated proteins; 3d, Heat map of different protein expression levels, low expression is shown in blue and high expression is shown in red; 3e, Unbiased clustering of 79544 cells from all nine samples revealed 8 cell clusters; 3f, The expression of SLC43A1 in smooth muscle cells (SMC) in the AD group was significantly higher than that in the CON group; 3g, Immunofluorescence staining (lower) of LAT3 (green) and α-SMA (red), and the nucleus was 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 (HASMC) treated with PDGF-BB (0, 20, 30, 40, 50, 60 ng / mL, treated for 24 h); n=4; 4b, qPCR analysis of SLC43A1 in HASMC treated with PDGF-BB (0, 20, 30, 40 ng / mL, treated for 24 h); n=4; 4c, qPCR analysis of SLC43A1 in HASMC treated with PDGF-BB (40 ng / mL, treated for 24 h) and CGA (0, 10, 20, 40, 8 4d, qPCR analysis of ACTA2 and OPP1 in HASMC treated with PDGF-BB (40 ng / mL, 24 h) and CGA (0, 10, 20, 40, 80 mM, 24 h); n=4; 4e, representative images and quantitative analysis of wound healing assay; n=3, scratch wound assay was performed on three groups of HASMC, and images were taken at 0 and 24 h after scratching (white line indicates wound edge). The migration area of ​​HASMC was analyzed from the average microscopic field of view. *p<0.05, **p<0.01, ***p<0.001.

[0031] Figure 5 : Figure 5a-5d, Branched chain amino acid (BCAA) levels in aorta tissue, serum samples, human aortic smooth muscle cells (HASMCs), and culture medium, respectively; n = 6; 5e, f, slc43a1 and α-SMAm RNA expression 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 aortas of each 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 by JASPER and HumanTFDB, potential binding sites of AR and SLC43A1 promoter sequence and corresponding mutations; 6d, e, Luciferase activity was detected by dual luciferase assay; n = 6. *p < 0.05, **p < 0.01, ***p < 0.001.

[0033] Figure 7 : Figure 7 a, 7b, Immunofluorescence staining (lower) of BCKDK / pBCKDHA (green) and α-SMA (red), nuclei were stained with DAPI (blue); 7c, BCKDK mRNA expression was determined by qRT-PCR, 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 DESCRIPTION

[0034] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are clearly and completely described. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0035] The present invention provides an application of chlorogenic acid in preventing and treating aortic dissection, which solves the technical problem that the current drugs for preventing and treating aortic dissection are very limited.

[0036] definition:

[0037] The term "inhibit" means to reduce by a measurable amount or to prevent entirely.

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

[0039] The term "patient" refers to a subject to whom the chlorogenic acid or salt thereof of the present invention is administered. 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, the patient is a rat, mouse, dog, human, or non-human primate, more typically a human.

[0040] The term "treatment" refers to therapeutic and preventive treatments, wherein the purpose is to inhibit or slow down (mitigate) undesirable physiological changes or conditions, such as the development or spread of cancer. For purposes of the present invention, useful or desired clinical results include, but are not limited to, alleviation of symptoms, weakening of disease extent, stabilization of disease state (i.e., no deterioration), delay or slowing of disease progression, improvement or alleviation of disease state, and relief (whether partial or complete), whether detectable or undetectable. "Treatment" may also mean an extension of survival compared to the expected survival without treatment. Those in need of treatment include those who have already suffered from the disease or condition and those who are susceptible to the disease or condition.

[0041] In the context of cancer, the term "treat" 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 reducing the number of cancer cells; and ameliorating one or more symptoms of associated diseases.

[0042] As used herein, the phrase "a salt thereof" refers to the salt form of a 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 is a detailed description through specific embodiments.

[0045] The chlorogenic acid (CGA) [drug number: Cat.#HY-N0055] involved in the examples was purchased from MedChemexpres; C57BL / 6 male mice (4 weeks old) were purchased from Henan Scobes Biotechnology Co., Ltd. All experiments were carried out in the SPF-level Animal Experiment Center of the First Affiliated Hospital of University of Science and Technology of China. The temperature of the breeding environment was controlled at 22-24°C. The experimental mice were free to eat and drink water. All animal experiments complied with the regulations of the Animal Ethics Committee of the First Affiliated Hospital of University of Science and Technology of China.

[0046] Data were expressed as mean ± standard error (M ± SEM) and statistically processed using Graphpad Prism 8 software. One-way ANOVA was used for sample comparison between two groups, and one-way ANOVA was used for comparison between more than two groups, followed by Newman-Keuls multiple comparison test. p < 0.05 was considered statistically significant.

[0047] Example 1: Establishment of AD animal model

[0048] Thirty-two 4-week-old C57 mice were randomly divided into three groups using statistical methods: CON control group, AD model group, and CGA treatment group. The mice in the CON group were given double distilled water for 28 days, and a micro-osmotic pump (Alzet, model 1003D) containing normal saline was implanted subcutaneously on the 28th day; the mice in the AD group were given double distilled water containing 0.4% BAPN (Sigma-aldrich, A3134, JAPAN) for 28 days, and then 1000 ng / kg / min angiotensin II (MCE, Cat, #HY-13948) was dripped through a micro-osmotic pump on the 28th day; the mice in the CGA group were given CGA 50 mg / kg intraperitoneally daily on the basis of AD modeling. During the experiment, all deaths and causes were recorded. After the experiment, all mice were euthanized and the aorta was dissected, and the aortic lesions were recorded under a stereomicroscope.

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

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

[0051] Transthoracic echocardiography was performed using a Visual Sonics Vevo 2100 system equipped with an MS400 probe (Visual Sonics). The width of the aortic arch was measured in mice at the level of the aorta. Anesthesia was induced by 3% isoflurane and confirmed by the absence of response to stimulation of one of the hind paws. During echocardiographic acquisition, isoflurane was reduced to 1.0–1.5% under thermostatic conditions and adjusted to maintain a heart rate in the range of 415–460 beats per minute. All mice recovered uneventfully after the test.

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

[0053] Example 3: Histochemical Analysis

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

[0055] like Figure 2 a: HE staining showed that red blood cells passed through the lumen and filled the separated vascular layer. Masson staining showed that the collagen fiber organization was disordered in the AD group. Electron microscopy staining showed that the elastic structure was obviously disordered and broken. The vascular structure of the CON group was intact. Although the aortic wall of the CGA group was thicker than that of the CON group, no obvious false lumen was formed, no obvious red blood cell infiltration was observed in the wall, and the collagen arrangement was disordered and the elastin fiber breakage was significantly reduced compared with the AD group.

[0056] Example 4: Immunohistochemical detection of the expression level of proteins related to HASMC phenotype conversion

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

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

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

[0060] Unbiased proteomic screening was performed on aorta samples from 3 mice in the CON, AD, and CGA groups. Overall, 44 proteins were differentially expressed (p<0.05, Log2(fold change)>2 or <-2) when comparing the data of the CON and AD groups, including 29 up-regulated proteins and 15 down-regulated 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(fold change)>2 or <-2), including 1 up-regulated protein and 24 down-regulated proteins ( Figure 3 b). Twenty-three overlapping proteins were found among the 29 up-regulated proteins (CON vs AD) and 24 down-regulated proteins (AD and CGA) ( Figure 3 cd). At the same time, the present invention analyzed the single-cell RNA sequencing data of aortic samples collected from 6 AD patients and 3 non-AD controls (GSE213740). The cells were divided into 8 major cell types ( Figure 3 e). The present invention pays special attention to the differentially expressed genes in the above overlapping proteins. We randomly sampled 4000 cells from the CON group and the AD group, 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 large neutral amino acid transporter small subunit 3 (LAT3) protein, which is located in the muscle plasma membrane and mediates the transport of neutral amino acids such as L-leucine, L-isoleucine and L-valine. To further verify the importance of LAT3, the present invention performed immunofluorescence staining to determine the expression of LAT3. The present invention's results clearly showed that LAT3 increased in the AD group, but was 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, the present invention used (human arterial smooth muscle cells) HASMC for in vitro experiments. α-SMA is a marker of SMCs that maintain a contractile phenotype, while secretory phosphoprotein 1 (SPP1) is a marker of a differentiated phenotype. The present invention used a concentration gradient (20, 30, 40, 50, 60 ng / mL) to determine the effect of PDGF-BB and found that after treatment with 40 ng / mL PDGF-BB, α -SMA had the lowest expression level, and SPP1 had the highest expression level ( Figure 4 a). Then, the present invention measured the effect of CGA using a concentration gradient (10, 20, 40, 80 μM) and co-treated with 40 ng / mL PDGF-BB. The present invention found that 40 μM CGA could reverse the effect of PDGF-BB on SMC phenotypic conversion to the greatest extent ( Figure 4 c). Meanwhile, CGA could reverse the increase of slc43a1 transcription induced by PDGF-BB in a concentration-dependent manner ( Figure 4 b, d). In addition, PDGF-BB-treated HASMCs showed increased migration, and co-treatment with CGA restored HASMCs to a stable state ( Figure 4 e) In conclusion, CGA reversed the PDGF-BB-induced SMC phenotypic transformation and increased slc43a1 transcription.

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

[0063] Example 7: CGA treats AD via 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 the levels of BCAAs in the aorta tissue of mice in the AD group were significantly increased compared with those in the CON group ( Figure 5 a), while CGA treatment reduced the levels of BCAAs in mouse aortic tissue and serum ( Figure 5 a, b). Consistent with the in vivo results, PDGF-BB treatment increased the content of BCAAs in SMCs, whereas CGA treatment reversed this effect ( Figure 5c). At the same time, the BCAA content in the cell culture medium of the PDGF-BB-treated wells was significantly lower than that of the CON group, and the BCAA content in the cell culture medium of the CGA-treated wells recovered, but there was no statistical significance ( Figure 5 d).

[0065] The above results indicate that the content of BCAA in SMCs may be a key factor affecting AD. Then, the present invention co-cultured cells with exogenous BCAA and PDGF-BB and CGA. PDGF-BB stimulation increased the expression of LAT3 in SMCs at the RNA and protein levels, and CGA treatment reversed this result. Incubating SMCs with BCAA while CGA treatment weakened the therapeutic effect of CGA, although this attenuation was not statistically significant ( Figure 5 e, g). Then the present 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, which was almost the same as that in the AD group ( Figure 5 g) Consistent with this, PDGF-BB stimulation can significantly reduce the expression of α-SMA, leaving the cells in a dedifferentiated state, while CGA treatment can restore the expression of α-SMA, but the addition of BCAA to CGA treatment can restore the cells to a dedifferentiated state. This means that BCAA together with CGA conferred and almost completely abolished the protective effect of CGA on HASMC ( Figure 5 f, g).

[0066] In order to further study whether the protective effect induced by CGA is related to LAT3, the present invention transfected HASMC with LAT3-OE plasmid to overexpress LAT3. The results showed that overexpression of LAT3 significantly reversed the protective effect of CGA on HASMC. On the basis of PDGF-BB stimulation, overexpression of LAT3 and phosphorylation of mTOR were further increased. At the same time, the expression of contractile markers in HASMC was statistically significantly reduced ( Figure 5 h). Consistent with the data from HASMC, in the AD group, upregulated pmTOR colocalized with the nucleus, whereas CGA treatment reversed this upregulation ( Figure 5 i). Taken together, these results suggest that CGA protects mice from AD via the LAT3-mTOR pathway.

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

[0068] Previous studies have reported that androgen receptor (AR) is a transcription factor that controls the transcription and expression of slc43a1. The present invention preliminarily found that CGA inhibited the upregulation of AR transcription levels caused by PDGF-BB stimulation of HASMC ( Figure 6 a), Immunohistochemistry results showed that compared with the AD group, the expression of AR in the nucleus of the CGA group was weakened ( Figure 6 b). It is speculated that the inhibitory effect of CGA on slc43a1 depends on the disappearance of AR.

[0069] The present invention uses JASPER and HumanTFDB to predict the binding site of AR on the slc43a1 promoter ( Figure 6 c). At the same time, AR-OE was constructed into the pcDNA3.1 vector. To determine the activity of the promoter, DNA of slc43a1, AR-OE and Renilla (for internal normalization) were transfected into HEK 293T cells (about 60-70% confluence) using Lipofectamine8000TM Transfection Reagent (Bio-Tech, China). Compared with the pGL3-Basic+pcDNA3.1 group, the luciferase activity of the slc43a1-WT+pcDNA3.1 group and the slc43a1-WT+AR group was significantly increased. Compared with the slc43a1-WT+pcDNA3.1 group, the luciferase activity of the slc43a1-WT+AR group was further increased ( Figure 6 d). These data indicate that AR can directly bind to the slc43a1 promoter and participate in the transcription of slc43a1. At the same time, the present invention also mutated the predicted binding site of AR on the human slc43a1 promoter. The results showed that the luciferase activity of the slc43a1-MT+pcDNA3.1 group was reduced compared with the slc43a1-WT+pcDNA3.1 group; similarly, the luciferase activity of the slc43a1-MT+AR group was also reduced compared with the slc43a1-WT+AR group. These results indicate that AR can bind to the TFBS of the slc43a1 promoter and regulate the transcription of slc43a1.

[0070] To further clarify the molecular mechanism of CGA inhibiting LAT3 expression, the present invention transfected slc43a1-WT or slc43a1-MT into HASMC and detected the promoter activity after CGA treatment. The mutation of slc43a1 and AR binding sites weakened the promoter activity of slc43a1 at the basal level, and also weakened the inhibitory effect of CGA on slc43a1 promoter activity ( Figure 6 e). The results showed that CGA inhibited the expression of slc43a1 at the transcriptional level by activating the AR pathway.

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

[0072] Previous studies have reported that the AD process is accompanied by BCAA metabolism disorders in SMCs, and the expression of branched-chain ketoacid dehydrogenase kinase (BCKDK) and the phosphorylation of branched-chain ketoacid dehydrogenase E1, alpha polypeptide (BCKDHA) are important rate-limiting enzymes in BCAA metabolism. Therefore, the present invention further explored the effect of CGA on the expression of BCKDK and BCKDHA. First, the present invention performed immunofluorescence staining on vascular sections of the CON group, AD group, and CGA group, and found that compared with the CON group, the expression of BCKDK and the phosphorylation of BCKDHA in the AD group were significantly upregulated, while CGA reversed this upregulation ( Figure 7 a, b). In order to study the changes in the expression of these two proteins, the present invention treated SMCs with the method described in the above experiment. Consistent with previous studies, PDGF-BB stimulation increased the expression of BDKDK and the phosphorylation of BCKDHA, and these increases meant that the metabolism of branched-chain amino acids was blocked, while functional incubation with CGA reversed this situation. Surprisingly, co-incubation of BCAA with PDGF-BB and CGA only alleviated the BCKDK downregulation induced by CGA treatment at the gene level, and did not alleviate the inhibitory effect of CGA on PDGF-BB damage, but further reduced the expression of BCKDK and the phosphorylation of BCKDHA. It was subsequently discovered that PDGF-BB stimulation led to increased BCKDK expression and increased BCKDHA phosphorylation in SMCs, while CGA treatment could reverse both increases. However, LAT3 overexpression had no effect on the changes in BCKDK and pBCKDHA ( Figure 7 c, d). These results suggest that treatment with CGA may affect BCKDK and, in parallel, the LAT3-mTOR pathway, but there is no connection between LAT3 and BCKDK.

[0073] In summary, the present invention has discovered for the first time the significant effect of chlorogenic acid in treating aortic dissection, providing a new idea and method for the treatment of aortic dissection. In addition, compared with traditional surgical operations and interventional treatments, the cost of using chlorogenic acid for treatment is relatively low, which reduces the economic burden on patients and also reduces the consumption of medical resources.

[0074] It should be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, the elements defined by the sentence "comprise a ..." do not exclude the existence of other identical elements in the process, method, article or device including the elements.

[0075] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. Use of chlorogenic acid or a pharmaceutically acceptable salt thereof in the preparation of a drug for preventing, inhibiting or treating aortic dissection.

2. The use according to claim 1, characterized in that The pharmaceutically acceptable salt of chlorogenic acid includes chlorogenic acid sodium salt, chlorogenic acid potassium salt or chlorogenic acid calcium salt.

3. Use of a pharmaceutical composition in the preparation of a drug for preventing, inhibiting or treating aortic dissection, characterized in that: The pharmaceutical composition comprises a therapeutically effective amount of chlorogenic acid or a pharmaceutically acceptable salt thereof.

4. The use according to claim 3, characterized in that The pharmaceutical composition comprises the combined use of chlorogenic acid or a pharmaceutically acceptable salt thereof and other drugs for treating aortic dissection.

5. The use according to claim 4, characterized in that The other drugs for treating aortic dissection are selected from at least one of antihypertensive drugs and beta-receptor blockers.

6. The use according to any one of claims 3 to 5, characterized in that: The drug for treating aortic dissection also includes pharmaceutically acceptable excipients.

7. Use of a pharmaceutical preparation in the preparation of a drug for preventing, inhibiting or treating aortic dissection, characterized in that: The pharmaceutical preparation comprises the pharmaceutical composition according to any one of claims 3-6.

8. The use according to claim 7, characterized in that The pharmaceutical preparation includes a solid dosage form, a semisolid dosage form, a liquid dosage form or a gas dosage form.

9. The use according to claim 8, characterized in that The solid dosage forms include powders, pills, tablets, and capsules; the semisolid dosage forms include ointments, suppositories, and gels; the liquid dosage forms include lotions, mixtures, solutions, and injections; and the gaseous dosage forms include aerosols and sprays.

10. The use according to claim 8, characterized in that The pharmaceutical preparation is in the form of tablets, capsules or injections.

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