Use of a natural inhibitor of the fabp4 gene, rosmarinic acid, for the preparation of a product for the treatment of atherosclerotic diseases

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to application of a natural inhibitor of FABP4 gene, rosmarinic acid, in preparation of products for treating atherosclerotic diseases. The application first discloses a direct regulation relationship between rosmarinic acid and FABP4 (fatty acid binding protein 4) gene, that is, rosmarinic acid can significantly down-regulate mRNA transcription level and protein expression level of the FABP4 gene, thereby providing a brand-new molecular mechanism explanation for known anti-atherosclerotic activity of rosmarinic acid. The application provides a new application of rosmarinic acid in preparation of drugs for treating and / or preventing atherosclerosis, and the application specifically delays atherosclerotic disease course progression by inhibiting FABP4 gene expression, thereby providing a natural lead compound and experimental basis for preparation of a new FABP4 targeted drug.

Description

[0001] This invention claims priority to earlier patent application No. 202611211191.5, filed on August 11, 2026, entitled "Application of a natural inhibitor of the FABP4 gene, rosmarinic acid, in the preparation of a product for treating atherosclerotic diseases". The entire contents of the above-cited application are incorporated herein by reference. Technical Field

[0002] This invention belongs to the field of biomedical technology, specifically relating to the application of rosmarinic acid, a natural inhibitor of the FABP4 gene, in the preparation of products for treating atherosclerotic diseases. Background Technology

[0003] Atherosclerosis (AS) is a chronic inflammatory and proliferative disease affecting the walls of large and medium-sized arteries throughout the body, initially triggered by lipid metabolism disorders. It is the common pathological basis for various high-risk cardiovascular events, including acute myocardial infarction and ischemic stroke. The course of atherosclerosis is regulated by multiple factors, including metabolic risk factors such as hypercholesterolemia, hypertension, and diabetes, endogenous pathological mechanisms such as inflammatory disorders and oxidative stress, and conditions such as poor dietary habits and genetic susceptibility, all of which contribute to the continuous progression of the disease. Current treatment protocols focus on managing risk factors and slowing plaque progression, lacking early warning and precise intervention strategies for unstable plaques.

[0004] Rosmarinic acid (RA) is a natural polyphenol with excellent antioxidant and anti-inflammatory activities, and has the advantage of multi-target regulation in the prevention and treatment of cardiovascular diseases. FABP4, as a key target regulating macrophage foaming and mediating lipid accumulation and inflammatory responses, participates in the progression of atherosclerosis. However, existing literature mostly verifies multiple cardiovascular protective pathways of rosmarinic acid or that natural products regulate lipid metabolism through the PPARγ-FABP4 signaling axis. It has not yet been systematically elucidated whether rosmarinic acid can directly target and regulate FABP4 gene transcription and protein expression. There is a lack of direct experimental evidence that rosmarinic acid acts on FABP4 to mediate foam cell formation in an ox-LDL-induced macrophage model. It is also unclear whether there is a direct binding effect between rosmarinic acid and FABP4 protein. The complete molecular mechanism by which rosmarinic acid regulates the upstream and downstream signaling networks of FABP4 and blocks the progression of atherosclerosis remains to be elucidated. It is difficult to clarify whether rosmarinic acid's inhibition of lipid deposition and alleviation of inflammatory responses depend on the FABP4 pathway, which restricts the in-depth development of rosmarinic acid as a candidate active substance targeting FABP4. Summary of the Invention

[0005] The purpose of this invention is to provide an application of rosmarinic acid, a natural inhibitor of the FABP4 gene, in the preparation of products for treating atherosclerotic diseases, thereby overcoming the shortcomings of the prior art and providing a new application of rosmarinic acid in the preparation of drugs for treating and / or preventing atherosclerosis. Specifically, this application delays the progression of atherosclerosis by inhibiting the expression of the FABP4 gene, thereby providing a natural lead compound and experimental basis for the preparation of novel FABP4-targeted drugs.

[0006] To achieve the above objectives, the technical solution of the present invention is as follows: The application of rosmarinic acid, a natural inhibitor of the FABP4 gene, in the preparation of products for the prevention and / or treatment of atherosclerotic diseases.

[0007] Furthermore, rosmarinic acid inhibits the expression level of the FABP4 gene.

[0008] Furthermore, a macrophage-derived foam cell model of atherosclerosis was constructed by inducing THP-1 cells and RAW264.7 cells with oxidized low-density lipoprotein (ox-LDL).

[0009] Furthermore, rosmarinic acid is an extract from the whole rosemary herb or from natural plants containing rosmarinic acid.

[0010] Furthermore, natural plants containing rosmarinic acid include perilla and salvia miltiorrhiza.

[0011] Furthermore, the purity of rosmarinic acid is greater than 98%.

[0012] Furthermore, the product is a drug, and the dosage form of the drug includes oral formulations and / or injectable formulations.

[0013] Furthermore, the drug includes rosmarinic acid and pharmaceutical adjuvants and / or excipients.

[0014] Furthermore, the concentration of rosmarinic acid in the product is 25-100 μmol / L.

[0015] Furthermore, the active ingredient in the product is rosmarinic acid.

[0016] The beneficial effects of this invention are: This invention reveals for the first time the direct regulatory relationship between rosmarinic acid and FABP4, a key target in atherosclerosis. Currently, there is no technological inspiration for rosmarinic acid to treat atherosclerosis by inhibiting FABP4 gene expression. This invention provides a novel molecular mechanism explanation for the anti-atherosclerotic effect of rosmarinic acid and also offers a new lead compound direction for drug development targeting FABP4.

[0017] Rosmarinic acid is a naturally occurring, water-soluble phenolic acid compound widely found in edible or medicinal plants such as rosemary, perilla, and salvia miltiorrhiza, with a long history of consumption and a good safety record. Compared with chemically synthesized FABP4 inhibitors (such as BMS-309403), the rosmarinic acid involved in this invention has lower toxicity, better biocompatibility, and greater safety for long-term use.

[0018] This invention confirms that rosmarinic acid can achieve a "one-target, multiple-point" regulatory effect by inhibiting FABP4, while improving vascular endothelial function, inhibiting abnormal proliferation of smooth muscle cells, reducing inflammatory response, reducing lipid accumulation and plaque formation, and has a comprehensive advantage in anti-atherosclerosis through multiple links and levels.

[0019] This invention provides in vitro cell experiments (THP-1 cells, RAW264.7 macrophages) and in vivo animal experiments (ApoE). - / - Complete pharmacological data from a mouse atherosclerosis model clearly demonstrate that rosmarinic acid can significantly inhibit FABP4 expression at both the mRNA and protein levels, and effectively reduce aortic plaque area, improve lipid profile and oxidative stress indicators. The technical solution has sufficient industrial applicability and reproducibility.

[0020] Rosmarinic acid has a mature extraction and purification process, with a wide range of raw material sources and low cost, making it suitable for large-scale industrial production and possessing good prospects for drug development and market competitiveness. Attached Figure Description

[0021] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0022] Figure 1 The image shows lipid deposition in foam cells induced by rosmarinic acid inhibition of ox-LDL. In the image, A shows Oil Red O staining of THP-1 cells and RAW264.7 cells, and B shows lipid deposition in foam cells. Figure 2To investigate how rosmarinic acid can alleviate the progression of atherosclerosis in ApoE- / - mice, the following diagrams are presented: A is a flowchart of the animal modeling process; B shows the weight change curves of mice in each group during a 16-week feeding period; C is a gross stained photograph of the entire aorta after dissection; D shows the percentage of the total aortic plaque area relative to the total vascular area in C; E shows lipid staining of frozen sections from the aortic root (arrows in the magnified image indicate lipid plaques); F shows the percentage of aortic root plaque area in E; G shows hematoxylin-eosin (HE) staining to observe vascular tissue structure and plaque morphology (arrows indicate atherosclerotic plaques); and H shows the quantitative percentage of the necrotic core area within the plaque relative to the total plaque area in G (the larger the necrotic core, the more unstable the plaque and the higher the cardiovascular risk). Figure 3 The diagram shows the regulatory effect of RA on FABP4 in macrophages. In the diagram, A shows the mRNA expression level of FABP4 in RAW264.7 cells detected by RT-qPCR, and B shows the protein expression of FABP4 detected by Western Blot. Figure 4 This represents the optimal conformation for Autodock docking proteins with small molecules. Detailed Implementation

[0023] Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be construed as limiting the scope of the invention. Specific conditions not specified in the embodiments are performed under conventional conditions or conditions recommended by the manufacturer. Components whose manufacturers are not specified are all commercially available conventional products.

[0024] Example 1 I. Experimental Section Rosmarinic acid is derived from the whole herb of rosemary (Rosmarinus officinalis L.), or other medicinal plants rich in rosmarinic acid such as perilla and salvia miltiorrhiza. There are no specific limitations on the preparation method of rosmarinic acid. Conventional extraction, separation and purification methods can be used. The purity of rosmarinic acid is ≥98%. It should be stored in a sealed container away from light and at low temperature for later use.

[0025] 1. Establishment of a macrophage-derived foam cell model and detection of lipid deposition. (1) Cell culture and induced differentiation Human monocyte cell line THP-1 in logarithmic growth phase was cultured in RPMI-1640 medium containing 10% fetal bovine serum, 100 U / mL penicillin, and 100 µg / mL streptomycin at 37°C and 5% CO2. THP-1 cells were treated with 100 ng / mL phorbol ester for 48 h to induce adherent differentiation into macrophages. Simultaneously, mouse macrophage cell line RAW264.7 was routinely cultured in high-glucose DMEM containing 10% fetal bovine serum. Further treatment was performed when cell confluence reached 70%–80%.

[0026] (2) Construction of foam cell model and drug intervention After discarding the original culture medium and gently washing with PBS, serum-free medium containing 100 µg / mL oxidized low-density lipoprotein was added to THP-1-derived macrophages and RAW264.7 cells, respectively. Cells were cultured for 24 h to induce intracellular lipid accumulation and construct a macrophage-derived foam cell model. A rosmarinic acid intervention group was also established, receiving 100 µM rosmarinic acid treatment 2 h before the addition of ox-LDL. The ox-LDL-only induction group served as the model control group, while the group without ox-LDL and the drug served as the blank control group.

[0027] (3) Qualitative observation of intracellular lipid deposition After induction and intervention, the degree of intracellular lipid deposition was assessed using Oil Red O staining. The culture medium was removed, cells were washed with PBS, and fixed with 4% paraformaldehyde at room temperature for 15 min. After washing with 60% isopropanol, cells were stained with Oil Red O working solution in the dark for 20 min. Cells were gently rinsed with distilled water, and images were acquired from at least five randomly selected fields of view under a microscope. Bound Oil Red O was extracted with isopropanol, and absorbance was measured at 510 nm for semi-quantitative analysis.

[0028] (4) Determination of intracellular total cholesterol and free cholesterol content After treatment, cells were collected, washed with PBS, centrifuged, and then fully lysed with cell lysis buffer. A tissue cell total cholesterol and free cholesterol assay kit was used, strictly following the instructions. A portion of the lysate supernatant was used to determine the total cholesterol content, while another portion was treated with a free cholesterol precipitant before determining the free cholesterol content. The protein concentration of the corresponding samples was determined using the BCA method to correct for lipid content; results are expressed as µg / mg protein.

[0029] 2. Construction and efficacy evaluation of apolipoprotein E knockout mouse model of atherosclerosis. (1) Establishment of experimental animals and models Select 6–8 week old male ApoE - / -Mice were randomly divided into a model group, a rosmarinic acid intervention group, and a positive control group after one week of SPF-grade environmental acclimatization, with 10 mice in each group; age-matched male wild-type mice (C57BL / 6J) served as blank controls. Except for the blank control group, which was given a normal maintenance diet, all other groups were fed a high-fat diet for 16 weeks to induce atherosclerotic lesions.

[0030] (2) Drug intervention program Starting from week 1 of high-fat feeding, the intervention group received 100 mg / kg / day of rosmarinic acid via gavage. The model group and the blank control group received an equal volume of solvent via gavage simultaneously, while the positive control group received either simvastatin or atorvastatin (5 mg / kg / day). The intervention lasted for 16 weeks, during which changes in body weight and food intake were monitored.

[0031] (3) Assessment of aortic plaque burden and area After the intervention, mice were anesthetized with isoflurane and euthanized by apical blood sampling. After perfusion of the left ventricle with pre-cooled PBS, the entire aorta (from the ascending aorta to the bifurcation of the iliac arteries) was meticulously dissected, and the extracorporeal fat and connective tissue were removed under a stereomicroscope. The aorta was longitudinally dissected and stained with Oil Red O under light-protected conditions: the entire aorta was immersed in 60% isopropanol for rinsing, then transferred to freshly prepared Oil Red O working solution for 1 h; subsequently, it was differentiated with 70% ethanol until the normal vessel wall was unstained and the plaque area was bright red. Images were captured using a high-resolution camera on a black background, and ImageJ software was used to calculate the percentage of positive red-stained aortic plaque area relative to the total aortic area to assess plaque burden.

[0032] (4) Histological analysis of plaque in cross-section of aortic root Tissue samples were harvested from the heart and the root of the ascending aorta, embedded in OCT scanners to prepare frozen sections with a thickness of 8–10 µm. Sections were collected via septa and stained with Oil Red O and hematoxylin-eosin to observe the lipid core area and intimal thickening of plaques within the aortic sinus. The intimal plaque area was measured using an image analysis system, and the ratio to the vascular lumen diameter was calculated to compare differences between groups.

[0033] 3. Verification of the molecular mechanism by which rosmarinic acid targets and regulates FABP4 (1) RT-qPCR detection of FABP4 mRNA expression level Based on the ox-LDL-induced RAW264.7 foam cell model, different concentrations of rosmarinic acid were used for intervention for 24 h. Total RNA was extracted from cells using the TRizol method, and RNA purity and concentration were detected by UV spectrophotometer. cDNA was synthesized using a reverse transcription kit. Mouse FABP4-specific primers (with GAPDH as the internal reference gene) were designed and synthesized, and real-time quantitative PCR was performed using the SYBR Green fluorescent dye method. The amplification program was: 95 °C pre-denaturation for 30 s; 95 °C for 5 s, 60 °C for 30 s, 40 cycles. 2 -ΔΔCt The relative expression level of FABP4 mRNA in each group was calculated using the method, and three independent experiments were conducted, with three replicates for each experiment.

[0034] (2) Western blot detection of FABP4 protein expression level RAW264.7 cells were lysed on ice using RIPA lysis buffer containing protease and phosphatase inhibitors after appropriate intervention to extract total protein. After quantification by BCA method, equal volumes of protein were separated by SDS-PAGE electrophoresis and transferred to PVDF membranes using wet transfer. After blocking with 5% skim milk at room temperature for 1 h, anti-FABP4 antibody and anti-GAPDH antibody were added, and the membranes were incubated overnight at 4°C. After thorough washing with TBST, HRP-labeled secondary antibodies of the corresponding species were added, and the membranes were incubated at room temperature for 1 h. After incubation with ECL chromogenic buffer, band images were acquired using a chemiluminescence imaging system. Grayscale analysis was performed using ImageJ software, and the ratio of the grayscale value of the target protein to that of the internal control protein was used as the relative protein expression level.

[0035] (3) Molecular docking simulates the binding of rosmarinic acid to FABP4 High-resolution crystal structures of human FABP4 protein (PDBID: e.g., 2NNQ) were downloaded from the Protein Data Bank database. The three-dimensional molecular structure of rosmarinic acid was plotted using ChemDraw, and the ligand conformation was optimized through energy minimization. Semi-flexible docking was performed using molecular docking software such as AutoDock Vina or Schrödinger, with a docking grid centered on the FABP4 endogenous fatty acid binding pocket. Based on binding energy scores, the binding modes of rosmarinic acid to FABP4 and the hydrogen bonding and hydrophobic interactions of key binding site residues were analyzed, and the results were visualized using PyMOL or Discovery Studio.

[0036] II. Performance Testing 1. Rosmarinic acid inhibits ox-LDL-induced lipid deposition in foam cells. Oil Red O staining shows ( Figure 1 A) Compared with the control group, ox-LDL (100 μg / ml) significantly increased the foaming level of THP-1 and RAW264.7 cells; while rosmarinic acid treatment significantly inhibited the increase of intracellular lipid droplets and reduced cell differentiation. The effect of rosmarinic acid on intracellular cholesterol content in ox-LDL-induced THP-1 and RAW264.7 foam cells was detected using the Amplex Red probe. Figure 1 As shown in Figure B, ox-LDL induction significantly increased intracellular TC and FC levels; rosmarinic acid intervention significantly reduced intracellular TC and FC levels and inhibited macrophage foaming. These results indicate that rosmarinic acid can alleviate ox-LDL-induced foam cell formation.

[0037] 2. Rosmarinic acid reduces ApoE. - / - Progression of atherosclerosis in mice ApoE gene knockout in 8-week-old males (ApoE) - / - Mice were fed a high-fat diet for 16 weeks to establish an atherosclerosis (AS) model. Observations were conducted at corresponding time points, and samples were collected at the 16-week endpoint. Figure 2 A).

[0038] Monitor the body weight change curve of mice during a 16-week feeding period to determine whether a high-fat diet and drug treatment affect mouse body weight. Figure 2 B). Compared with the NC group, the HFD group showed a significant increase in arterial plaque formation, covering 32.58±6.29% of the total aortic area. However, the L-RA and H-RA groups showed a significant reduction in aortic plaque, with plaque areas decreasing by 26.97±5.92% and 20.98±2.54%, respectively. Figure 2 (C and 2D).

[0039] To further investigate the distribution of lipid plaques in the aortic root, Oil Red O staining was performed on the mouse aortic valve. Compared with the control group (NC), the plaque area in the high-fat diet (HFD) group was significantly larger, accounting for 43.21±4.32% of the aortic root. In contrast, the plaque area in the L-RA and H-RA treatment groups was significantly reduced, decreasing by 29.89±2.25% and 24.19±2.89%, respectively. Figure 2 E and 2F). Quantitative analysis of the percentage of necrotic core area within the plaque was performed using HE staining. Results showed that the necrotic core area in the HFD group was significantly larger than that in the control group, while the necrotic core area in the RA treatment group was significantly smaller (E and 2F). Figure 2 G and 2H).

[0040] 3. Rosmarinic acid (RA) targets and inhibits FABP4 expression. To clarify the targeted regulation of FABP4 expression by rosmarinic acid, the effect of rosmarinic acid on FABP4 expression in ox-LDL-induced RAW264.7 cells was verified by RT-qPCR and Western blot. The relative expression level of FABP4 mRNA was detected by RT-qPCR. Compared with the normal control group (CN), FABP4 mRNA expression in the ox-LDL model group was significantly upregulated. After intervention with gradient concentrations of 25, 50, and 100 μmol / L rosmarinic acid, the expression level of FABP4 mRNA decreased in a concentration-dependent manner. Figure 3 A). FABP4 protein expression was detected by Western blotting, with GAPDH as an internal control. The quantitative protein analysis results were consistent with the trend at the gene level. ox-LDL stimulation significantly increased FABP4 protein expression, and treatment with different concentrations of rosmarinic acid inhibited FABP4 protein upregulation in a concentration-dependent manner. Figure 3 B). The results showed that rosmarinic acid could significantly inhibit the mRNA and protein expression of FABP4 in macrophages.

[0041] Molecular docking was used to predict the binding mode of rosmarinic acid to FABP4. Based on the three-dimensional structure of mouse FABP4 predicted by AlphaFold, molecular docking was first performed using AutoDock-Vina to predict the optimal binding conformation of rosmarinic acid in the FABP4 binding pocket. Figure 4 Nine binding modes were generated during the docking calculations. The binding affinity and root mean square deviation (RMSD) of these modes relative to the optimal mode are shown in Table 1.

[0042] Table 1. Results of AutoDock Vina molecular docking between rosmarinic acid and mouse FABP4.

[0043] As shown in Table 1, among all docking conformations, Mode 1 exhibits the lowest binding free energy (-8.5 kcal / mol), making it the predicted optimal binding conformation. The binding energies of the other eight conformations range from -8.2 to -7.4 kcal / mol, all being negative, indicating that the binding of rosmarinic acid and FABP4 is thermodynamically spontaneous and favorable. Considering both binding energy and RMSD, Mode 1 was determined to be the energy-optimal and conformationally reasonable binding mode and was chosen as the initial conformation for subsequent molecular dynamics simulations.

[0044] To evaluate the binding free energy of the rosmarinic acid-FABP4 complex in molecular dynamics simulations, the overall MM-GBSA binding free energy of the rosmarinic acid-mouse FABP4 complex was analyzed (Table 2).

[0045] Table 2. Free energy of MM-GBSA binding of rosmarinic acid to mouse FABP4 complex.

[0046] As shown in Table 2, the total binding free energy (ATOTAL) of rosmarinic acid and FABP4 calculated by MM-GBSA is -26.37 kcaV / mol, which is a large negative value. This indicates that the formation of the complex between the two in solution is a thermodynamically favorable process, further verifying the reliability of the molecular docking prediction.

[0047] The gas phase energy (AGGAS) contributed -66.43 kcal / mol, which was the primary energy source driving the binding. Van der Waals interactions (AVDWAALS, -35.27 kcal / mol) and electrostatic interactions (AEEL, -31.16 kcal / mol) contributed roughly equally, indicating that the hydrophobic stacking and van der Waals contacts between the phenolic acid backbone of rosmarinic acid and the hydrophobic cavity of FABP4, as well as the hydrogen bond network formed between the phenolic hydroxyl / carboxyl groups and the polar residues of the protein, together constituted the main driving force for binding.

[0048] The polar solvation energy (AEGB, +45.86 kcal / mol) is a large positive value, reflecting the need to overcome the desolvation energy barrier when transferring the ligand from the aqueous phase to the protein binding pocket, which is the main adverse factor for the binding free energy. The nonpolar solvation energy (AESURF, -5.81 kcal / mol) originates from the reduction in exposed hydrophobic surface area and has a weak promoting effect on binding. The solvation energy (AGSOLV, +40.05 kcal / mol) partially offsets the contribution of the gas phase energy, resulting in a final net binding free energy of -26.37 kcal / mol.

[0049] In summary, the recognition mechanism of rosmarinic acid by FABP4 exhibits a typical pattern of "gas-phase energy-driven, solvation energy-offset". Notably, electrostatic interactions account for a significant proportion of the gas-phase energy (-31.16 kcalV / mol), suggesting that the polar structure of rosmarinic acid, rich in phenolic hydroxyl and carboxyl groups, forms a strong polar interaction network within the FABP4 binding pocket. This may help explain its superior binding affinity compared to most endogenous long-chain fatty acids (which primarily rely on hydrophobic interactions). These calculations provide a quantitative energetic basis for the molecular mechanism of rosmarinic acid as a natural ligand of FABP4.

[0050] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. The use of rosmarinic acid, a natural inhibitor of the FABP4 gene, in the preparation of products for the prevention and / or treatment of atherosclerotic diseases.

2. The application as described in claim 1, characterized in that, The rosmarinic acid inhibits the expression level of the FABP4 gene.

3. The application as described in claim 1, characterized in that, The atherosclerotic disease was modeled using macrophage-derived foam cells constructed from THP-1 cells induced by oxidized low-density lipoprotein (ox-LDL) and RAW264.7 cells.

4. The application as described in claim 1, characterized in that, The rosmarinic acid is an extract of the whole rosemary herb or a natural plant containing rosmarinic acid.

5. The application as described in claim 4, characterized in that, The natural plants containing rosmarinic acid include perilla and salvia miltiorrhiza.

6. The application as described in claim 1, characterized in that, The purity of the rosmarinic acid is greater than 98%.

7. The application as described in claim 1, characterized in that, The product is a drug, and the dosage form of the drug includes oral preparations and / or injectable preparations.

8. The application as described in claim 7, characterized in that, The drug includes rosmarinic acid and pharmaceutical adjuvants and / or excipients.

9. The application as described in claim 1, characterized in that, The concentration of rosmarinic acid in the product is 25-100 μmol / L.

10. The application as described in claim 1, characterized in that, The active ingredient in the product is rosmarinic acid.

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