Pharmaceutical composition for preventing endothelial activation

Abstract

The present invention belongs to the technical field of pharmaceutical compositions, and provides a pharmaceutical composition for preventing endothelial activation, l-CDL (L-corydaline) or a pharmaceutically acceptable salt thereof is used as an active ingredient, the l-CDL enhances the expression of nitric oxide synthase by inhibiting the transcriptional activity of DDIT3 (DNA damage induced transcriptase 3), and the expression of the nitric oxide synthase is enhanced by inhibiting the transcriptional activity of the DDIT3. Therefore, endothelial cell activation induced by ox-LDL is inhibited, and vasodilation is improved; the present invention provides a pharmaceutical composition for preventing endothelial activation, the composition uses l-CDL or a pharmaceutically acceptable salt thereof as an active ingredient, and effectively enhances the expression of nitric oxide synthase by inhibiting the transcriptional activity of DDIT3, thereby significantly inhibiting the endothelial cell activation induced by ox-LDL, and further improving the vasodilation function. A new target spot and a treatment strategy are provided for preventing cardiovascular diseases.

Description

Technical Field

[0001] The invention belongs to the technical field of pharmaceutical compositions, in particular to a pharmaceutical composition for preventing endothelial activation. Background Art

[0002] Atherosclerosis is the main pathological basis of many cardiovascular diseases, which begins with endothelial cell activation. The existing technology has paid attention to the potential of the traditional Chinese medicine Corydalis yanhusuo in treating cardiovascular diseases, however, its specific molecular mechanism is not fully understood.

[0003] Network pharmacology and molecular docking analysis showed that l-CDL has a strong binding affinity to atherosclerosis-related targets; the cellular level further verified that l-CDL has the effect of inhibiting ox-LDL-induced endothelial cell activation, reducing the levels of inflammatory and aging markers, and upregulating eNOS levels; animal experimental studies also confirmed that l-CDL can promote endothelium-dependent vasodilation under both physiological and pathological conditions.

[0004] To this end, those skilled in the art have proposed a pharmaceutical composition for preventing endothelial activation, aiming to explore the effect of l-CDL (levorotatory corydaline), a tetrahydroberberine-type isoquinoline alkaloid extracted or synthesized from Corydalis yanhusuo, on endothelial cell activation and its mechanism. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides a pharmaceutical composition for preventing endothelial activation, comprising: l-CDL (levorotatory corydaline) or a pharmaceutically acceptable salt thereof as an active ingredient.

[0006] Preferably, l-CDL inhibits the transcriptional activity of DDIT3 (DNA damage-induced transcript 3) and enhances the expression of nitric oxide synthase (eNOS), thereby inhibiting ox-LDL (oxidized low-density lipoprotein)-induced endothelial cell activation and improving vasodilation.

[0007] Preferably, the pharmaceutical composition is used to prepare a drug for preventing or treating endothelial cell activation, atherosclerosis and related cardiovascular diseases.

[0008] A method for preventing cardiovascular disease comprises: administering the above-mentioned pharmaceutical composition to an individual in need thereof.

[0009] Preferably, the cardiovascular disease includes, but is not limited to, atherosclerosis, endothelial dysfunction, vascular inflammation or vascular aging.

[0010] The use of the above-mentioned pharmaceutical composition for preventing endothelial activation in the preparation of drugs for preventing cardiovascular diseases.

[0011] Compared with the prior art, the present invention has the following beneficial effects:

[0012] 1. Provided is a pharmaceutical composition for preventing endothelial activation, which uses l-CDL (levorotatory corydaline) or a pharmaceutically acceptable salt thereof as an active ingredient, and effectively enhances the expression of nitric oxide synthase (eNOS) by inhibiting the transcriptional activity of DDIT3 (DNA damage-induced transcript 3), thereby significantly inhibiting ox-LDL (oxidized low-density lipoprotein)-induced endothelial cell activation, thereby improving vasodilation function, and providing a new target and treatment strategy for preventing cardiovascular diseases.

[0013] 2. The pharmaceutical composition described in the present invention is widely used in the preparation of drugs for preventing or treating endothelial cell activation, atherosclerosis and related cardiovascular diseases; by administering the pharmaceutical composition to individuals in need, the symptoms of cardiovascular diseases such as atherosclerosis, endothelial dysfunction, vascular inflammation and vascular aging can be alleviated, thereby improving the quality of life of patients.

[0014] 3. The present invention demonstrates through experiments the remarkable effect of the l-CDL pharmaceutical composition in preventing cardiovascular diseases; the experimental results show that the pharmaceutical composition can inhibit endothelial cell activation, alleviate cardiovascular disease symptoms such as atherosclerosis, and provide a new option for the treatment of cardiovascular diseases; at the same time, the experimental results of the present invention also provide strong support for the further research and development of the l-CDL pharmaceutical composition, and has broad market application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 Schematic diagram of l-CDL of the present invention alleviating ox-LDL-induced HUVECs endothelial cell activation; wherein, (A) in the ox-LDL model, the effect of different concentrations of l-CDL or ox-LDL on cell viability and the effect of different concentrations of l-CDL on cell viability under ox-LDL conditions; (B) l-CDL reduces the mRNA levels of IL-6, IL-8, ICAM-1, TNF-α, NF-κB, JUN, FOS and PTGS2 in the ox-LDL model; (C) L-CDL reduces the protein levels of VCAM-1 and e-selectin in the ox-LDL model; (D) L-CDL eliminates the increase in SA-β-gal activity induced by ox-LDL; (E) L-CDL eliminates the increase in p21 level induced by ox-LDL; compared with the control group, **P<0.01, **P<0.001, ****P<0.0001; compared with the ox-LDL group, # P<0.01, ## P<0.05, ### P<0.001, ####P<0.0001,n=5.

[0016] Figure 2 Schematic diagram of the improvement of ox-LDL-induced endothelial dysfunction by l-CDL of the present invention, wherein (A) L-CDL improves ox-LDL-induced endothelial barrier dysfunction; (B) L-CDL restores NO level and fluorescence intensity in the ox-LDL model; (C) L-CDL upregulates the mRNA and protein levels of eNOS in the ox-LDL model; compared with the control group, **P<0.01, ****P<0.0001; compared with the ox-LDL group, ## P<0.05, ### P<0.001, #### P<0.0001,n=5.

[0017] Figure 3 Schematic diagram of the improvement of endothelium-dependent vasodilation by l-CDL of the present invention under physiological and pathological conditions; wherein, (A) schematic diagram of vascular tension and treatment time; (B) vasodilation effect of I-CDL, ATV, ox-LDL+I-CDL and ox-LDL+ATV on rat aorta; (C) vasodilation effect of I-CDL in the L-EC group and L-NAME group; *P<0.05, ****P<0.0001, #### P<0.0001, n=3.

[0018] Figure 4 Schematic diagram of the present invention showing that l-CDL improves endothelial cell activation by inhibiting DDIT3 transcription and increasing eNOS expression, wherein (A) volcano plot and Venn diagram show the DEGs between ox-LDL group and Control group and between ox-LDL+l-CDL group and ox-LDL group; (B) KEGG pathway enrichment analysis of 422 DEGs; (C) heat map of 11 DEGs related to the "lipid and atherosclerosis" pathway; (D) PPI network of 11 DEGs related to the "lipid and atherosclerosis" pathway; (E) DDIT3 expression is increased in the blood of AS patients; (F) L-CDL reduces the transcriptional activity of DDIT3 in the ox-LDL model; (G) dual luciferase reporter gene experiment confirms the negative regulatory effect of DDIT3 on eNOS promoter activity; (H) L-CDL restores eNOS expression inhibited by DDIT3 in the ox-LDL model; compared with the control group, ****P<0.0001; compared with the ox-LDL group, ### P<0.001, #### P<0.0001,n=5. DETAILED DESCRIPTION

[0019] The following embodiments of the present invention are described in further detail in conjunction with the accompanying drawings and examples. The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention.

[0020] Embodiment: The present invention provides a pharmaceutical composition for preventing endothelial activation, comprising: l-CDL (levorotatory corydaline) or a pharmaceutically acceptable salt thereof as an active ingredient; l-CDL is a tetrahydroberberine-type isoquinoline alkaloid extracted or synthesized from Corydalis yanhusuo; wherein, l-CDL enhances the expression of nitric oxide synthase (eNOS) by inhibiting the transcriptional activity of DDIT3 (DNA damage-induced transcript 3), and this mechanism of action further inhibits endothelial cell activation induced by ox-LDL (oxidized low-density lipoprotein) and effectively improves vasodilation function.

[0021] Figure 1 The results show that l-CDL can reduce ox-LDL-induced endothelial cell activation of HUVECs (human umbilical vein endothelial cells); Part (A) of the figure shows the effect of different concentrations of l-CDL on cell viability in the ox-LDL model, indicating that l-CDL has the effect of protecting cell viability; Part (B) of the figure shows that l-CDL reduces the levels of multiple inflammation-related mRNAs in the ox-LDL model, including IL-6, IL-8, ICAM-1, TNF-α, NF-κB, JUN, FOS and PTGS2; Part (C) of the figure shows that l-CDL reduces the protein levels of VCAM-1 and E-selectin in the ox-LDL model; Parts (D) and (E) of the figure show that l-CDL eliminates the increase in SA-β-gal activity and p21 levels induced by ox-LDL, respectively, indicating that l-CDL has anti-aging and anti-inflammatory effects.

[0022] Figure 2 The results show that l-CDL can improve ox-LDL-induced endothelial barrier dysfunction; (A) shows that l-CDL can improve ox-LDL-induced endothelial barrier dysfunction; (B) shows that l-CDL restored the NO level and fluorescence intensity in the ox-LDL model; (C) shows that l-CDL upregulated the mRNA and protein levels of eNOS in the ox-LDL model, further confirming that l-CDL improves endothelial function by enhancing eNOS expression.

[0023] Figure 3The effect of l-CDL in improving endothelium-dependent vasodilation under physiological and pathological conditions is demonstrated; Part (A) of the figure shows a schematic diagram of vascular tension and treatment time; Part (B) of the figure shows that l-CDL has a significant vasodilatory effect on rat aorta, and the effect is better than the commonly used vasodilator ATV (atorvastatin); Part (C) of the figure shows that in the presence of L-NAME (N-nitro-L-arginine methyl ester), the vasodilatory effect of l-CDL is inhibited, further confirming that l-CDL improves vasodilation by increasing NO levels.

[0024] Figure 4 The mechanism by which l-CDL improves endothelial cell activation by inhibiting DDIT3 transcription and increasing eNOS expression is shown; Part (A) of the figure shows the differentially expressed genes (DEGs) between the ox-LDL group and the control group and between the ox-LDL+l-CDL group and the ox-LDL group through volcano plots and Venn diagrams; Part (B) of the figure shows the KEGG pathway enrichment analysis of 422 DEGs, and the results show that genes related to the "lipid and atherosclerosis" pathway are significantly enriched; Part (C) of the figure shows 11 DEGs related to the "lipid and atherosclerosis" pathway s heat map; Part (D) shows the protein-protein interaction (PPI) network of these 11 DEGs; Part (E) shows that DDIT3 expression is increased in the blood of AS (atherosclerosis) patients; Part (F) shows that l-CDL reduces the transcriptional activity of DDIT3 in the ox-LDL model; Part (G) confirms the negative regulatory effect of DDIT3 on eNOS promoter activity by dual luciferase reporter gene experiment; Part (H) shows that l-CDL restores eNOS expression inhibited by DDIT3 in the ox-LDL model.

[0025] Based on the above mechanism of action and experimental verification, a method for preventing cardiovascular diseases comprises: administering the above pharmaceutical composition to an individual in need thereof, wherein the cardiovascular diseases include but are not limited to atherosclerosis, endothelial dysfunction, vascular inflammation or vascular aging.

[0026] The l-CDL screening method comprises the following steps:

[0027] Step 1: Screening of Corydalis yanhusuo related targets:

[0028] The components of Corydalis yanhusuo were searched through the TCMSP platform, and the active ingredients and their protein targets that met the DL value ≥ 0.18 and oral bioavailability (OB) ≥ 30% were screened out;

[0029] Supplement known targets of active compounds that have not been predicted based on currently published technical literature;

[0030] Standardize the protein target information of compounds in the UniPort protein database;

[0031] Step 2: Screening of atherosclerosis-related targets:

[0032] Using diseases related to atherosclerosis as keywords, we explored potential targets in the GeneCards and Disgenet databases, and supplemented them with first-line Western medicine targets for regulating dyslipidemia from the DRUGBANK database.

[0033] Set the screening threshold to screen out potential targets for atherosclerosis and merge and remove duplicates;

[0034] Step 3: Construction of Corydalis yanhusuo-atherosclerosis target PPI network:

[0035] Intersect the targets related to Corydalis yanhusuo and those related to atherosclerosis and draw a Venn diagram;

[0036] Submit the intersection targets to the STRING database to construct a protein-protein interaction (PPI) network model and screen the core targets;

[0037] Step 4: Enrichment analysis of Yanhusuo components-atherosclerosis target functions and pathways:

[0038] The Corydalis yanhusuo-atherosclerosis targets were entered into the Metascape platform for GO and KEGG enrichment analysis, and the Compound-target-pathway (CTP) network was constructed;

[0039] Step 5: Molecular docking verification:

[0040] Obtain the pdb structure file of the target protein from the PDB protein structure database and the mol2 file of the core monomer from the TCMSP database;

[0041] Molecular docking was performed using molecular docking software to dock the top 10 core targets in the CTP network with the core monomers;

[0042] The binding strength and activity of the target and active compound were evaluated according to the Docking Score value, and the Levo-Corydalmine (l-CDL) monomer with the best binding activity to the atherosclerosis-related target was screened out.

[0043] The embodiments of the present invention are further described in detail below in conjunction with the accompanying drawings and application examples.

[0044] Application example: Verification of the effect of l-CDL pharmaceutical composition in preventing cardiovascular disease

[0045] The present invention provides a pharmaceutical composition for preventing endothelial activation, wherein the main active ingredient is l-CDL (levorotatory corydaline) or a pharmaceutically acceptable salt thereof; in order to verify the actual effect of the pharmaceutical composition, we conducted the following application example experiments:

[0046] 1. Experimental materials:

[0047] 1) l-CDL pharmaceutical composition (the active ingredient is l-CDL or a pharmaceutically acceptable salt thereof);

[0048] 2) Experimental vector: human umbilical vein endothelial cells, rats (used to simulate cardiovascular disease models);

[0049] 3) Reagents and instruments: ox-LDL (oxidized low-density lipoprotein), cell culture medium, microplate reader, microscope, and image analysis software.

[0050] 2. Experimental methods:

[0051] 1) Animal grouping and model establishment:

[0052] ① Gently separate the rat aorta and place each group of aortic segments (2 mm) in Krebs solution, with 3 segments in each group.

[0053] ② The aortas with intact endothelium were divided into l-CDL group, l-CDL+ox-LDL group, ATV group, and ATV+ox-LDL group. They were stabilized in Krebs solution or Krebs solution containing ox-LDL for 30 minutes and stimulated with phenylephrine (PE) for about 20 minutes to induce maximum contraction. -10 to 10 -5 Drug concentration gradients of M were added into the chamber every 2 minutes, and their vasodilatory effects were recorded.

[0054] ③ In the L-NAME group (pretreated with L-NAME for 15 min) or -EC group (endothelium was mechanically removed), PE was used to induce maximum contraction and after reaching equilibrium, acetylcholine (Ach) was given to determine whether the drug exerted an endothelium-dependent effect.

[0055] 2) Index detection:

[0056] ① Detect the vascular tension index of rat vascular rings;

[0057] 3) Cell experiments:

[0058] ①Isolate and culture rat aortic endothelial cells;

[0059] ②The cells were divided into ox-LDL group, ox-LDL+l-CDL group and control group;

[0060] ③Add corresponding concentrations of ox-LDL and l-CDL to each group of cells at different time points to observe cell morphology, proliferation and senescence;

[0061] ④ Detect cell viability using an ELISA instrument and observe cell morphology changes using a microscope and image analysis software.

[0062] 4) Data analysis:

[0063] ① Conduct statistical analysis on the experimental data and compare the differences between the experimental group and the control group;

[0064] ② Display the experimental results in the form of charts, including cell viability curves, biochemical index change charts, etc.

[0065] 3. Experimental results:

[0066] 1) Animal experiment results:

[0067] ① Whether in normal physiological environment or ox-LDL-induced pathological state, l-CDL shows excellent vasodilatory function, far exceeding the efficacy of the control drug ATV. In addition, the vasodilatory effect of l-CDL does depend on endothelial function.

[0068] 2) Cell experiment results:

[0069] ① Under the action of ox-LDL, the morphology of endothelial cells changes significantly and the cell viability decreases;

[0070] ②After adding l-CDL, the cell morphology gradually recovered and the cell viability was significantly improved;

[0071] ③Compared with the control group, the proliferation ability of cells in the l-CDL group was enhanced and the apoptosis rate was reduced. 3) Description of the drawings:

[0073] ① Figure 1 The results showed that l-CDL can alleviate ox-LDL-induced HUVECs endothelial cell activation, which proved the protective effect of l-CDL on endothelial cells;

[0074] ② Figure 2 The effect of l-CDL in improving ox-LDL-induced endothelial barrier dysfunction was demonstrated, further confirming the improving effect of l-CDL on endothelial function;

[0075] ③ Figure 3 and Figure 4 By demonstrating the mechanism by which l-CDL improves endothelium-dependent vasodilation under physiological and pathological conditions, and improves endothelial cell activation by inhibiting DDIT3 transcription and increasing eNOS expression, we provide a theoretical basis for l-CDL to prevent cardiovascular diseases.

[0076] As can be seen from the above, through the experimental verification of this application example, we have proved the significant effect of the l-CDL pharmaceutical composition in preventing cardiovascular diseases; the pharmaceutical composition can inhibit endothelial cell activation, alleviate cardiovascular disease symptoms such as atherosclerosis, and provide a new option for the treatment of cardiovascular diseases; at the same time, the experimental results of this application example also provide strong support for the further research and development of the l-CDL pharmaceutical composition.

[0077] The embodiments of the present invention are provided for the purpose of illustration and description. Although the embodiments of the present invention have been shown and described above, it can be understood that the above embodiments are exemplary and cannot be understood as limitations of the present invention. Ordinary technicians in this field can change, modify, replace and modify the above embodiments within the scope of the present invention.

Claims

1. A pharmaceutical composition for preventing endothelial activation, characterized in that: include: 1-CDL or a pharmaceutically acceptable salt thereof is used as an active ingredient.

2. A pharmaceutical composition for preventing endothelial activation according to claim 1, characterized in that: in, l-CDL inhibits ox-LDL-induced endothelial cell activation and improves vasodilation by inhibiting the transcriptional activity of DDI T3 and enhancing the expression of nitric oxide synthase.

3. A pharmaceutical composition for preventing endothelial activation according to claim 2, characterized in that: Used for preparing drugs for preventing or treating endothelial cell activation, atherosclerosis and related cardiovascular diseases.

4. A method for preventing cardiovascular disease, characterized in that: include: Administering the pharmaceutical composition of claim 1 or 2 to an individual in need thereof.

5. A method for preventing cardiovascular disease according to claim 4, characterized in that: The cardiovascular disease includes, but is not limited to, atherosclerosis, endothelial dysfunction, vascular inflammation or vascular aging.

6. Use of the pharmaceutical composition for preventing endothelial activation according to claim 4 in the preparation of drugs for preventing cardiovascular diseases.

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