Application of IAA in preparation of medicine for preventing and treating atherosclerosis

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to application of IAA in preparation of a medicine for preventing and treating atherosclerosis. Atherosclerosis is caused by excessive deposition of cholesterol, and IAA can significantly reduce formation of aorta and aortic sinus lipid plaques of atherosclerosis model mice by adjusting monocyte chemotactic and adhesion functions (specifically, down-regulating expression of chemotactic factors CCL2 and CXCL1, receptors CCR1 and CCR2, adhesion molecules ICAM1 and the like). An injection preparation is preferably selected as a medicine dosage form, the intervention dosage of IAA is 50 mg / kg, and the intervention time is 15 weeks. The new application of IAA in prevention and treatment of atherosclerosis is explored for the first time, the action mechanism of IAA is clear, the intervention effect is remarkable, and a new target spot and thought are provided for developing safe and effective medicines and means for preventing and treating atherosclerosis.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the application of an IAA in the preparation of drugs for the prevention and treatment of atherosclerosis. Background Technology

[0002] Cardiovascular disease causes more deaths worldwide than any other type of disease. AS is a complex, multifactorial disease, and its pathogenesis is not yet fully understood. Studying the pathogenesis of AS is of great significance for finding therapeutic targets for coronary heart disease.

[0003] Indole-3-acetic acid (IAA) is a small molecule compound containing an indole ring structure. It was first extracted and identified by Dutch scientists in 1934 from oat coleoptiles and human urine. Initially known as a core plant auxin, it uses L-tryptophan as its initial precursor and is converted into an indole-3- acetic acid.

[0004] Pyruvate (IPA) and other pathways are synthesized in plants, regulating cell elongation, organ morphogenesis, and stress adaptation, making it a key signaling molecule for plant growth and development. Further research has revealed that IAA is not exclusive to plants: In humans, gut microbiota such as Bacteroides ovalis and Bifidobacteria can metabolize tryptophan from the host's diet to produce IAA. This gut-derived IAA can enter the systemic circulation via the portal vein, activating pathways such as the aryl hydrocarbon receptor (AHR) as signaling molecules, participating in immune regulation (e.g., regulating macrophage polarization and suppressing inflammation) and metabolic homeostasis, and even being associated with the progression of diseases such as atherosclerosis. In contrast, the human body can only produce a very small amount of IAA through tryptophan metabolism, mainly relying on gut microbiota synthesis and plant-based food intake, ultimately being metabolized by the liver and excreted in urine. Today, IAA has become an important molecule spanning plant physiology, microbiology, and medicine, and is one of the key targets for studying the interaction between diet, gut microbiota, and host health, as well as the mechanisms of metabolic diseases. Currently, there are no reports, either domestically or internationally, on the benefits of IAA in the prevention and treatment of atherosclerosis. Summary of the Invention

[0005] To address the aforementioned problems, this invention proposes the application of IAA in the preparation of drugs for the prevention and treatment of atherosclerosis, thereby more precisely resolving the issues raised in the background section.

[0006] This invention is achieved through the following technical solution:

[0007] This invention proposes the application of IAA in the preparation of drugs for the prevention and treatment of atherosclerosis, which is caused by excessive cholesterol deposition.

[0008] Preferably, IAA

[0009] By regulating the chemotaxis and adhesion function of monocytes, lipid plaque formation in animal models of atherosclerosis was reduced.

[0010] Preferably, the IAA regulates the chemotaxis and adhesion function of monocytes by downregulating the expression levels of chemokines CCL2 and CXCL1, chemokine receptors CCR1 and CCR2, and adhesion molecule ICAM1.

[0011] Preferably, the dosage form of the drug is an injectable formulation.

[0012] Preferably, the injectable formulation is an intraperitoneal injection formulation.

[0013] Preferably, the intervention dose of IAA is 50 mg / kg.

[0014] Preferably, the continuous intervention period of IAA is 15 weeks.

[0015] Preferably, the drug further comprises excipients selected from one or more of water for injection, physiological saline, phosphate-buffered saline (PBS), polyethylene glycol 400, and glycerin, for maintaining the stability and injection safety of the IAA.

[0016] Compared with the prior art, the present invention provides an application of IAA in the preparation of drugs for the prevention and treatment of atherosclerosis, which has the following beneficial effects:

[0017] First discovery of a new application for IAA: This invention is the first to confirm that IAA can be used to prevent and treat atherosclerosis, filling the research gap in the application of IAA in the field of cardiovascular diseases and expanding its medical application value.

[0018] The mechanism of action is clearly defined and targets key pathological processes: This invention clarifies that IAA exerts its preventive and therapeutic effects by regulating monocyte chemotaxis and adhesion, with specific targets including...

[0019] CCL2, CXCL1, CCR1, CCR2, ICAM1

[0020] These molecules are key regulators of early inflammatory responses in atherosclerosis, with clearly defined targets and clinical translational potential.

[0021] Significant intervention effect: Animal experiments confirmed that after 15 weeks of continuous intraperitoneal injection of 50 mg / kg IAA, the amount of lipid deposition in the aorta and aortic sinus of atherosclerotic model mice was significantly reduced (p < 0.05), and there was no adverse effect on the mice's body weight, fasting blood glucose, blood lipids and other basic indicators, indicating that it has good safety. Attached Figure Description

[0022] To more clearly illustrate the technical solution of this application, the drawings used in the embodiments will be briefly introduced below. Obviously, for those skilled in the art, other drawings can be obtained based on these drawings without any creative effort.

[0023] Figure 1 This is a schematic diagram illustrating the effect of IAA intervention on plaques in various groups of AS mice, provided in an embodiment of this application.

[0024] Figure 2 A schematic diagram illustrating the effect of IAA on baseline indicators in AS mice, provided as an embodiment of this application.

[0025] Figure 3 This is a schematic diagram illustrating the effect of IAA on monocyte adhesion and chemotaxis in AS mice, provided in an embodiment of this application.

[0026] Figure 4 A schematic diagram illustrating the effect of IAA on the inflammatory response of LPS-stimulated THP-1 cells, provided in an embodiment of this application.

[0027] Figure 5 A schematic diagram illustrating the effect of IAA on LPS-induced chemotaxis and adhesion molecule levels in THP-1 cells, as provided in an embodiment of this application.

[0028] Figure 6 A schematic diagram illustrating the effect of IAA on the expression levels of CCR2 and ICAM1 in THP-1 cells, provided as an embodiment of this application.

[0029] Figure 7 A schematic diagram illustrating the chemotaxis and adhesion of IAA to LPS-induced THP-1 cells, provided as an embodiment of this application. Detailed Implementation

[0030] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0031] The core of this invention is to verify the preventive and therapeutic effects and mechanism of indole-3-acetic acid (IAA) on atherosclerosis through animal and cell experiments, and to clarify its application value in the preparation of drugs for the prevention and treatment of atherosclerosis. The invention is described in detail below through specific embodiments.

[0032] Example 1: Experimental Materials and Reagents

[0033] 1. Experimental animals: Twenty 6-8 week old SPF-grade male ApoE- / - mice were purchased from the Nanjing Model Animal Resource Bank and housed in an SPF-grade animal room with a temperature of 22±2℃, humidity of 50±5%, and a 12-hour light-dark cycle, with free access to food and water.

[0034] 2. Cell line: Human monocytic leukemia cell line THP-1, purchased from the Cell Bank of the Chinese Academy of Sciences.

[0035] 3. Main reagents:

[0036] Indole-3-acetic acid (IAA, purity ≥98%): purchased from Sigma-Aldrich.

[0037] High-fat feed (containing 0.5% cholesterol and 21% fat): purchased from Nanjing Xietong Biotechnology Co., Ltd.

[0038] Lipopolysaccharide (LPS, E. coli O111:B4): purchased from Sigma-Aldrich;

[0039] Oil Red O staining kit, HE staining kit, and Masson's staining kit: purchased from Beijing Solarbio Science & Technology Co., Ltd.

[0040] Blood lipid test kit (total cholesterol, triglycerides, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol): purchased from Nanjing Jiancheng Bioengineering Institute;

[0041] TNF-α, IL-6, CCL2 ELISA kit: purchased from R&D Systems;

[0042] Anti-mouse CD11b-PC5.5, CD115-APC, CCR1-FITC, CCR2-PE, ICAM1-PB450 antibodies, and anti-human CCR2-APC, ICAM1-FITC antibodies: purchased from BD Biosciences.

[0043] RNA extraction kit, reverse transcription kit, and qPCR kit: purchased from Takara Corporation;

[0044] Transwell chamber (8μm pore size): purchased from Corning.

[0045] All other routine reagents were of analytical grade and purchased from Sinopharm Chemical Reagent Co., Ltd.

[0046] 4. Main instruments:

[0047] Stereo microscope (Olympus SZX16): purchased from Olympus (China) Co., Ltd.;

[0048] Optical microscope (Olympus BX53): purchased from Olympus (China) Co., Ltd.;

[0049] Flow cytometer (BD LSR Fortessa): purchased from BD Biosciences;

[0050] qPCR instrument (ABI StepOnePlus): purchased from Applied Biosystems;

[0051] Microplate reader (Bio-Tek Synergy H1): Purchased from Bio-Tek Instruments Co., Ltd.;

[0052] Cryostat (Leica CM1950): Purchased from Leica Microsystems (Shanghai) Co., Ltd.

[0053] Example 2: Effects of IAA on plaque formation in Atherosclerotic mouse model (ApoE- / - mice)

[0054] 1. Animal grouping and treatment:

[0055] Twenty 6-8 week old SPF-grade male ApoE- / - mice were acclimatized for one week and then randomly divided into two groups (n=10):

[0056] Model group (Vehicle)

[0057] Group 1: Feeded with a high-fat diet and injected intraperitoneally with an equal volume of physiological saline daily for 15 consecutive weeks;

[0058] IAA intervention group (IAA group): fed a high-fat diet and received intraperitoneal injection of 50 mg / kg IAA (dissolved in physiological saline) daily for 15 consecutive weeks.

[0059] During the experiment, the mice’s food intake and weight were recorded weekly, and fasting blood glucose was tested every 4 weeks.

[0060] 2. Sample collection and processing:

[0061] After 15 weeks of intervention, mice were fasted for 12 hours and anesthetized by intraperitoneal injection of 1% sodium pentobarbital (50 mg / kg). Blood was collected through the orbital venous plexus, and plasma was separated by centrifugation at 3000 r / min for 15 min for the detection of blood lipids and cytokines. The mice were then sacrificed, and the aorta (from the ascending aorta to the bifurcation of the abdominal aorta) and aortic sinus were dissected for pathological staining and molecular biological detection.

[0062] 3. Gross Oil Red O staining of the aorta:

[0063] The isolated aorta was rinsed with PBS to remove surrounding connective tissue, longitudinally cut open, and laid flat on a glass slide. It was fixed with 4% paraformaldehyde for 30 min and washed three times with distilled water. Oil Red O staining solution was added for 30 min, and it was differentiated with 60% isopropanol until the background was colorless. It was washed three times with distilled water. Hematoxylin counterstaining was performed for 5 min, and the aorta was rinsed with tap water to regain its blue color. The images were taken under a stereomicroscope, and ImageJ software was used to quantitatively analyze the percentage of lipid deposition area in the total aorta area.

[0064] 4. Pathological sections and staining of the aortic sinus:

[0065] The isolated aortic sinus tissue was embedded in tissue freezing medium, frozen at -20°C, and then continuously sectioned (8 μm thick) using a cryostat and mounted on a glass slide.

[0066] Oil Red O staining: Sections were dried at room temperature for 30 min, fixed with 4% paraformaldehyde for 10 min, and washed 3 times with distilled water; stained with Oil Red O solution for 15 min, differentiated with 60% isopropanol for 30 s, and washed 3 times with distilled water; counterstained with hematoxylin.

[0067] After 2 minutes, rinse with tap water and the color returns to blue; mount with glycerol gelatin, photograph under an optical microscope, and use ImageJ software to quantitatively analyze the percentage of lipid plaque area in the aortic sinus lumen area;

[0068] HE staining: Sections were dried at room temperature for 30 min, fixed with 4% paraformaldehyde for 10 min, and washed 3 times with distilled water; hematoxylin staining was applied for 5 min, followed by rinsing with tap water to restore blue color; eosin staining was applied for 30 s, followed by rinsing with distilled water 3 times.

[0069] Next, the arterial wall was dehydrated with graded ethanol, cleared with xylene, mounted with neutral resin, and the pathological structural changes were observed under an optical microscope.

[0070] Masson staining: Sections were dried at room temperature for 30 min, fixed with 4% paraformaldehyde for 10 min, and washed 3 times with distilled water; stained with hematoxylin for 5 min, rinsed with tap water to regain blue color; stained with Ponceau S and Acid Fuchsin for 10 min, and washed 3 times with 1% glacial acetic acid; differentiated with phosphomolybdic acid for 5 min, and washed 3 times with 1% glacial acetic acid; stained with aniline blue for 5 min, and washed 3 times with 1% glacial acetic acid; dehydrated with graded ethanol, cleared with xylene, mounted with neutral resin, and observed under an optical microscope for collagen fiber deposition.

[0071] 5. Blood lipid and fasting blood glucose tests:

[0072] Following the instructions of the lipid testing kit, the levels of total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C) in plasma were measured using an ELISA reader; fasting blood glucose was measured using the glucose oxidase method.

[0073] 6. Statistical Analysis:

[0074] Experimental data were statistically analyzed using SPSS 26.0 software. Quantitative data were expressed as mean ± standard deviation (x ± s). Independent samples t-tests were used for comparisons between groups, and p < 0.05 was considered statistically significant.

[0075] 7. Experimental Results:

[0076] 7.1 Food intake, body weight, and fasting blood glucose:

[0077] like Figure 2 As shown in A, B, and C, during the 15-week intervention period, there were no significant differences in the average daily food intake, weight change, and fasting blood glucose levels between the IAA group and the Vehicle group (p > 0.05), suggesting that IAA had no adverse effects on the basal nutritional intake and glucose metabolism of mice.

[0078] 7.2 Blood lipid levels:

[0079] As shown in Figure 2D, there were no statistically significant differences in plasma TC, TG, LDL-C, and HDL-C levels between the two groups of mice (p > 0.05), suggesting that IAA does not play a role in preventing and treating atherosclerosis by regulating blood lipid levels.

[0080] 7.3 Plaque formation in the aorta and aortic sinus:

[0081] like Figure 1 As shown in B and C, Oil Red O staining results indicated that, compared to the Vehicle group, the IAA group mice had a significantly lower percentage of gross lipid deposition area in the aorta (Vehicle group: 18.2±3.5% vs IAA group: 10.5±2.1%, p<0.05), and the percentage of lipid plaque area in the aortic sinus was also significantly lower (Vehicle group: 22.6±4.2% vs IAA group: 13.1±2.8%, p<0.05); HE staining showed that the Vehicle...

[0082] The aortic sinus wall of the mice in the first group was significantly thickened, and a large number of foam cells and lipid accumulation were visible under the intima. In contrast, the aortic sinus wall thickening of the IAA group was reduced, and the number of foam cells was significantly decreased. Masson staining showed that collagen fiber deposition in the aortic sinus plaques of the IAA group was slightly increased compared with that of the Vehicle group, suggesting that plaque stability may be improved.

[0083] The above results indicate that continuous intraperitoneal injection of 50 mg / kg IAA for 15 weeks can significantly reduce the formation of atherosclerotic plaques in ApoE- / - mice, without adverse effects on the mice's basic physiological indicators.

[0084] Example 3: Study on the mechanism of IAA regulating chemotaxis and adhesion function of ApoE- / - mouse monocytes

[0085] 1. PCR array analysis of the aortic AS pathway:

[0086] Aortic tissues (n=5) were collected from mice in the Vehicle and IAA groups. Total RNA was extracted using an RNA extraction kit, and cDNA was synthesized by reverse transcription. The expression differences of chemotaxis, adhesion, and inflammation-related genes in the aortic tissues of the two groups of mice were analyzed using a qPCR array kit (containing 84 AS-related genes) according to the instructions of the qPCR instrument. The relative gene expression levels were calculated using the 2-ΔΔCt method. The fold change in gene expression was represented by Log2(IAA group / Vehicle group), with Log2>1 indicating upregulation and Log2<-1 indicating downregulation.

[0087] 2. Plasma chemokine detection:

[0088] Take the plasma samples isolated in Example 2 (n=10), and follow the instructions of the CCL2 and CXCL1 ELISA kit to detect the concentration of CCL2 and CXCL1 in the plasma using an enzyme-linked immunosorbent assay (ELISA) reader.

[0089] 3. Detection of peripheral blood mononuclear cell phenotype and molecular expression:

[0090] One mL of fresh mouse peripheral blood was collected, anticoagulated with EDTA, and peripheral blood mononuclear cells (PBMCs) were separated by density gradient centrifugation. The PBMCs were washed twice with PBS and the cell concentration was adjusted to 1×10⁶ / mL. Anti-mouse CD11b-PC5.5, CD115-APC, CCR1-FITC, CCR2-PE, and ICAM1-PB450 antibodies were added and incubated at 4°C in the dark for 30 min. After washing twice with PBS, the proportion of monocytes (CD11b+CD115+) in the PBMCs and the positive expression rates of CCR1, CCR2, and ICAM1 on the surface of monocytes were detected by flow cytometry.

[0091] 4. Statistical Analysis:

[0092] Same as in Example 2, p < 0.05 indicates that the difference is statistically significant.

[0093] 5. Experimental Results:

[0094] 5.1 Differences in gene expression in aortic tissue:

[0095] PCR array analysis results as follows Figure 3 As shown in Figure A, compared with the Vehicle group, the expression of five genes related to monocyte chemotaxis and adhesion was significantly downregulated in the aortic tissue of the IAA group mice. These genes were: chemokines, phosphatases, and phosphatases.

[0096] The presence of CCL2 (Log2 = -1.83), CXCL1 (Log2 = -1.56), chemokine receptor CCR1 (Log2 = -1.32), and adhesion-related genes Sell (Log2 = -1.28) and Spp1 (Log2 = -1.15) suggests that IAA may inhibit monocyte chemotaxis and adhesion by downregulating the expression of these genes.

[0097] 5.2 Plasma chemokine concentrations:

[0098] like Figure 3 As shown in B, the concentrations of CCL2 (Vehicle group: 45.6±8.2 pg / mL vs IAA group: 28.3±6.5 pg / mL, p<0.05) and CXCL1 (Vehicle group: 32.1±5.8 pg / mL vs IAA group: 19.5±4.2 pg / mL, p<0.05) in the plasma of mice in the IAA group were significantly lower than those in the Vehicle group, consistent with the results of the PCR array.

[0099] 5.3 Peripheral blood mononuclear cell phenotype and molecular expression:

[0100] Flow cytometry analysis results as follows Figure 3 As shown in C and D, there was no significant difference in the proportion of monocytes (CD11b+CD115+) in the PBMCs of the two groups of mice (Vehicle group: 8.2±1.5% vs IAA group: 7.8±1.2%, p>0.05); however, the positive rates of CCR1 (Vehicle group: 38.9±4.6% vs IAA group: 26.0±3.8%, p<0.05), CCR2 (Vehicle group: 35.7±5.2% vs IAA group: 22.5±4.1%, p<0.05), and ICAM1 (Vehicle group: 83.9±6.5% vs IAA group: 78.5±5.8%, p<0.05) on the surface of monocytes were significantly lower in the IAA group than in the Vehicle group.

[0101] The above results indicate that IAA

[0102] It can reduce plasma chemokine concentration by downregulating the expression of chemokines (CCL2, CXCL1) in aortic tissue and inhibit the expression of chemokine receptors (CCR1, CCR2) and adhesion molecules (ICAM1) on the surface of peripheral blood monocytes, thereby inhibiting the chemotactic and adhesion functions of monocytes. This may be the core mechanism by which it reduces the formation of atherosclerotic plaques.

[0103] Example 4: Effects of IAA on LPS-induced inflammatory response and chemotactic adhesion function of THP-1 cells

[0104] 1. Cell culture and grouping:

[0105] THP-1 cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin in a cell culture incubator at 37°C and 5% CO2. Cells in the logarithmic growth phase were harvested, and the cell concentration was adjusted to 1 × 10⁶ / mL. The cells were then seeded in 6-well or 96-well plates and divided into 4 groups.

[0106] Control group (CON group): Added an equal volume of culture medium, without any treatment;

[0107] LPS group: Add 1000 ng / mL LPS and incubate for 6 h;

[0108] LPS+IAA group: After pretreatment with 1000 ng / mL IAA for 12 h, 1000 ng / mL LPS was added and cultured for 6 h.

[0109] IAA group: Add 1000 ng / mL IAA and incubate for 18 h.

[0110] 2. Optimal intervention time selection:

[0111] To determine the optimal time for LPS to induce inflammatory response in THP-1 cells and the intervention effect of IAA, different intervention time points (0, 3, 6, 9, 12 h) were set. The concentration of TNF-α in the cell culture medium of each group was detected by ELISA, and the peak time of TNF-α was taken as the optimal intervention time.

[0112] 3. Inflammatory factor detection:

[0113] The concentrations of TNF-α and IL-6 in the cell culture medium of each group were detected according to the instructions of the TNF-α and IL-6 ELISA kit.

[0114] 4. Detection of chemotactic and adhesion molecule mRNA expression:

[0115] Total RNA was extracted from cells in each group using an RNA extraction kit, and cDNA was synthesized by reverse transcription. GAPDH was used as an internal reference gene, and qPCR was used to detect the mRNA expression levels of CCR1, CCR2, CCL2, CXCL1, and ICAM1. The qPCR primer sequences are shown in Table 1. Detection of chemokine and adhesion protein expression:

[0116] ELISA assay: The protein concentration of CCL2 in the cell culture medium of each group was detected according to the instructions of the CCL2 ELISA kit.

[0117] Flow cytometry detection: Collect cells from each group, adjust the concentration to 1×106 / mL, add anti-human CCR2-APC and ICAM1-FITC antibodies, and incubate at 4℃ in the dark for 30 min; after washing twice with PBS, detect the positive expression rate of CCR2 and ICAM1 by flow cytometry.

[0118] 5. Transwell chemotaxis experiment:

[0119] Logarithmic growth phase THP-1 cells were collected and their concentration adjusted to 2×10⁵ / mL. 600 μL of culture medium containing 100 ng / mL CCL₂ was added to the lower chamber, and 200 μL of cell suspension was added to the upper chamber. LPS and IAA were added according to the grouping. After culturing in a 37℃, 5% CO₂ incubator for 4 h, the Transwell chamber was removed, and the unmigrated cells in the upper chamber were wiped off with a cotton swab. The cells were fixed with 4% paraformaldehyde for 10 min and stained with crystal violet for 15 min. The number of migrating cells was counted in 5 random fields of view under an optical microscope, and the average value was taken.

[0120] 6. Cell adhesion experiment:

[0121] 96-well plates were coated with 10 μg / mL fibronectin and incubated overnight at 4°C. After washing three times with PBS, 1% bovine serum albumin was added for blocking for 2 h. After washing three times with PBS, 100 μL / well of THP-1 cell suspension adjusted to 1×10⁵ / mL was added, and LPS and IAA were added according to the grouping. After culturing at 37°C and 5% CO₂ for 1 h, the plates were washed three times with PBS to remove non-adhered cells. After staining with 0.5% crystal violet for 15 min, the plates were washed three times with PBS. After dissolving the crystal violet with 10% acetic acid, the absorbance (OD value) at 570 nm was measured using a microplate reader. The higher the OD value, the stronger the cell adhesion ability.

[0122] 7. Statistical Analysis:

[0123] Same as the embodiment

[0124] 2. One-way ANOVA was used for comparisons among multiple groups, and LSD-t test was used for pairwise comparisons between groups. p < 0.05 was considered statistically significant.

[0125] 8. Experimental Results:

[0126] 9.1 Screening for the optimal intervention time:

[0127] like Figure 4 As shown in Figure A, after LPS stimulation, the concentration of TNF-α in the THP-1 cell culture medium gradually increased over time, reaching a maximum of 6 h.

[0128] The concentration of TNF-α reached its peak at 6 hours (412.5±35.6 pg / mL) and then gradually decreased. IAA pretreatment significantly reduced the concentration of TNF-α, and the intervention effect was most obvious at 6 hours (LPS group: 412.5±35.6 pg / mL vs LPS+IAA group: 205.3±28.4 pg / mL, p<0.0001). Therefore, 6 hours was determined to be the optimal intervention time.

[0129] 9.2 Expression of inflammatory factors:

[0130] like Figure 4 As shown in B and C, compared with the CON group, the concentrations of TNF-α and IL-6 in the cell culture medium of the LPS group were significantly increased (p < 0.0001); compared with the LPS group, the concentrations of TNF-α and IL-6 in the LPS+IAA group were significantly decreased (p < 0.001); there was no significant difference between the IAA group and the CON group (p > 0.05), suggesting that IAA can inhibit the LPS-induced inflammatory response of THP-1 cells.

[0131] 9.3 Expression of chemotactic and adhesion molecules mRNA:

[0132] As shown in Figures 5A and 5B, compared with the CON group, the mRNA expression levels of CCR1, CCR2, CCL2, CXCL1, and ICAM1 in the LPS group were significantly increased (p < 0.0001); compared with the LPS group, the mRNA expression levels of the above genes in the LPS+IAA group were significantly decreased (p < 0.01); there was no significant difference between the IAA group and the CON group (p > 0.05).

[0133] 9.4 Expression of chemotactic and adhesion proteins:

[0134] ELISA results as follows Figure 5 As shown in Figure C, the concentration of CCL2 protein in the cell culture medium of the LPS group was significantly higher than that of the CON group (p < 0.0001), while the concentration of CCL2 protein in the LPS+IAA group was significantly lower than that in the LPS group (p < 0.001); the flow cytometry results are as follows. Figure 6 As shown in B and C, the positive rates of CCR2 (76.8±5.2%) and ICAM1 (78.4±6.1%) in the LPS group were significantly higher than those in the CON group (12.3±2.1% and 10.5±1.8%, respectively, p<0.0001), while the positive rates of CCR2 (67.7±4.8%) and ICAM1 (60.0±5.5%) in the LPS+IAA group were significantly lower than those in the LPS group (p<0.05).

[0135] 9.5 Cell chemotaxis and adhesion ability:

[0136] The results of the Transwell chemotaxis assay are shown in Figure 7A. The number of migrating cells in the LPS group (215.6±18.5) was significantly higher than that in the CON group (32.5±5.8, p<0.0001), while the number of migrating cells in the LPS+IAA group (128.3±12.6) was significantly lower than that in the LPS group (p<0.001). The results of the cell adhesion assay are shown in Figure 7A. Figure 7 As shown in B, the OD value of the LPS group (0.68±0.07) was significantly higher than that of the CON group (0.15±0.03, p<0.0001), and the OD value of the LPS+IAA group (0.42±0.05) was significantly lower than that of the LPS group (p<0.001).

[0137] The above cell experiment results further confirm that IAA

[0138] It can inhibit the chemotaxis and adhesion ability of monocytes by downregulating the expression of chemokines (CCL2, CXCL1), chemokine receptors (CCR1, CCR2) and adhesion molecules (ICAM1), while reducing the inflammatory response, thereby exerting an anti-atherosclerotic effect.

[0139] Example 5: Preparation of Injectable Formulation

[0140] Taking intraperitoneal injection preparations as an example, the formulation for preparing an injection containing IAA is as follows:

[0141] Active ingredient: IAA 50mg;

[0142] Solvent: 1 mL of physiological saline;

[0143] Stabilizer: 0.05 mL glycerol.

[0144] Preparation method:

[0145] 1. Take 1 mL of sterile saline, add 50 mg of IAA, and stir magnetically until completely dissolved;

[0146] 2. Add 0.05 mL of glycerin and continue stirring for 5 minutes until well mixed;

[0147] 3. Sterilize by filtration using a 0.22μm microporous membrane;

[0148] 4. Dispense into 1mL sterile ampoules, seal, and the product is ready.

[0149] The injectable formulation has a pH of 7.2-7.4, which meets the quality standards for injectables. It can be used for intraperitoneal injection, exhibits good stability, and shows no significant decrease in content when stored at 4°C for 6 months. Figures 1-7 As shown, this invention is proposed in one embodiment.

[0150] Finally, it should be noted that the basic concepts have been described above. Obviously, for those skilled in the art, the detailed disclosure above is merely illustrative and does not constitute a limitation of this specification. Although not explicitly stated herein, those skilled in the art may make various modifications, improvements, and corrections to this specification. Such modifications, improvements, and corrections are suggested in this specification, and therefore remain within the spirit and scope of the exemplary embodiments of this specification. Furthermore, this specification uses specific terms to describe embodiments of this specification. For example, "an embodiment," "one embodiment," and / or "some embodiments" refer to a feature, structure, or characteristic associated with at least one embodiment of this specification. Therefore, it should be emphasized and noted that "an embodiment," "one embodiment," or "an alternative embodiment" mentioned twice or more in different locations in this specification do not necessarily refer to the same embodiment. In addition, certain features, structures, or characteristics in one or more embodiments of this specification can be appropriately combined. Moreover, unless expressly stated in the claims, the order of processing elements and sequences, the use of numbers and letters, or other names described in this specification are not intended to limit the order of the processes and methods of this specification.

[0151] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. The application of an IAA in the preparation of drugs for the prevention and treatment of atherosclerosis, characterized in that, The atherosclerosis is caused by excessive cholesterol deposition.

2. The application of the IAA according to claim 1 in the preparation of drugs for preventing and treating atherosclerosis, characterized in that, The IAA reduces lipid plaque formation in animal models of atherosclerosis by regulating the chemotaxis and adhesion function of monocytes.

3. The application of the IAA according to claim 2 in the preparation of drugs for preventing and treating atherosclerosis, characterized in that, The IAA regulates monocyte chemotaxis and adhesion by downregulating chemokines. The expression levels of CCL2, CXCL1, chemokine receptors CCR1 and CCR2, and adhesion molecule ICAM1.

4. The application of the IAA according to claim 1 in the preparation of drugs for preventing and treating atherosclerosis, characterized in that, The drug is in the form of an injectable preparation.

5. The application of the IAA according to claim 4 in the preparation of drugs for preventing and treating atherosclerosis, characterized in that, The injectable preparation is an intraperitoneal injection preparation.

6. The application of the IAA according to claim 1 in the preparation of drugs for preventing and treating atherosclerosis, characterized in that, The intervention dose of the IAA is 50 mg / kg.

7. The application of the IAA according to claim 6 in the preparation of drugs for preventing and treating atherosclerosis, characterized in that, The IAA's continuous intervention period is 15 weeks.

8. According to claim 1 The application of the aforementioned IAA in the preparation of drugs for the prevention and treatment of atherosclerosis is characterized in that... The drug also includes excipients selected from one or more of water for injection, physiological saline, phosphate-buffered saline (PBS), polyethylene glycol 400, and glycerin, to maintain the stability and injection safety of the IAA.

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