Application of honeysuckle glycoside in preparation of medicine for treating vascular injury

Abstract

The invention relates to the technical field of medicines, in particular to application of honeysuckle glycoside in preparation of a medicine for treating vascular injury. The invention provides application of honeysuckle glycoside in preparation of a medicine for treating vascular injury. Experimental research finds that the lonicera japonica has a remarkable curative effect on treating the vascular injury induced by high glucose, and the action pathway of the lonicera japonica is a Ca < 2 + > pathway. The research shows that the honeysuckle glycoside serving as an important active component of the honeysuckle is wide in source distribution, easy to obtain and low in cost, is expected to become a novel traditional Chinese medicine preparation for treating the vascular injury, and can play an important role in occurrence and development of clinical treatment of the vascular injury in the future.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technology, and in particular to the application of lonicerin in the preparation of drugs for treating vascular injury. Background Art

[0002] Diabetes is characterized by metabolic disorders of sugars, lipids, proteins, etc., and its prevalence rate increases year by year. The complications of diabetes can damage multiple tissues and organs. Among them, the risk of cardiovascular diseases in diabetic patients is 8 times higher than that of normal people, and 70%-80% of diabetic patients die from cardiovascular diseases. Vascular endothelium is an important part of the cardiovascular system, which affects many aspects of vascular function, including pressure, flow, permeability, angiogenesis, and blood flow changes. Vascular endothelial injury is the pathological basis for diabetes-induced cardiovascular diseases. Vascular endothelial injury will lead to vascular dysfunction, and further lead to cardiovascular lesions such as atherosclerosis, cardiac hypoxia and ischemia, myocardial infarction, cardiac dysfunction, and heart failure. At present, there are few drugs for treating diabetic vascular injury. Therefore, it is very urgent to discover effective drugs for treating diabetic vascular injury. The discovery of new drugs will provide options for clinical use of drugs for diabetic vascular injury, relieve the economic burden brought by diabetes to patients and society, and has important social significance.

[0003] Under certain conditions (such as high glucose, hypoxia, oxidative stress, inflammation, abnormal fluid shear stress, etc.), endothelial cells will transform into mesenchymal cells (EndMT). The phenotypic transformation of endothelial cells involves the loss of endothelial-specific markers, including platelet endothelial cell adhesion molecule-1 (CD31), Tie1, Tie2, vascular endothelial cadherin (VE-cadherin), von Willebrand factor (VWF), etc., and the acquisition of mesenchymal markers, including α-smooth muscle actin (α-SMA), fibroblast specific protein (FSP1), N-cadherin (N-Cadherin), and vimentin (Vimentin), etc. During this process, endothelial cells lose their original functions, such as the ability to form blood vessels, the ability to inhibit thrombin formation, the ability to bind lectin, and the ability to uptake low-density lipoprotein cholesterol, and instead acquire some functions of mesenchymal cells, such as contractility, invasiveness, migratory ability, and collagen secretion characteristics. Studies have shown that under diabetic conditions, hyperglycemia and inflammatory factors can induce EndMT. These endothelial cells that have undergone EndMT not only lose the functions of endothelial cells, but also promote the production of extracellular matrix, promote the thickening and fibrosis of the vascular wall, trigger vascular remodeling, increase the rigidity and thickness of blood vessels, thereby affecting hemodynamics and vascular function. In addition, endothelial cells that have undergone EndMT can secrete a variety of cytokines and chemokines, further exacerbating the local inflammatory response. This inflammatory response not only damages vascular endothelium, but also promotes the occurrence and development of atherosclerosis.

[0004] Lonicerin is a natural compound extracted from plants of the genus Lonicera. Lonicera, also known as honeysuckle, is a medicinal plant widely used in traditional Chinese medicine, with effects such as clearing heat and detoxifying, dissipating swelling and nodules. Lonicerin is one of its main active ingredients, with anti-inflammatory and immunomodulatory effects. It has been previously reported that lonicerin has anti-inflammatory and anti-apoptotic activities in lipopolysaccharide (LPS)-induced acute lung injury, and plays an important role in reducing macrophage inflammation in ulcerative colitis mice. In addition, it has also been reported that lonicerin has an anti-inflammatory effect on murine arthritis. In this study, we used diabetes as a model to induce vascular endothelial injury to verify the therapeutic effect of lonicerin on it, providing a new therapeutic drug for the treatment of diabetic vascular injury in clinical work. Summary of the Invention

[0005] The object of the present invention is to provide the application of lonicerin in the preparation of drugs for treating vascular injury. The present invention has found through research that lonicerin has a therapeutic effect on various diabetic complications, but its effect on diabetic-induced vascular endothelial injury has not been reported so far. Through experimental methods such as immunofluorescence, Western blot, and qPCR, it was detected that lonicerin significantly reduces diabetic-induced vascular endothelial injury in aspects such as vascular calcification, vascular fibrosis, vascular inflammation, and the process of endothelial-mesenchymal transition (EndMT) of vascular endothelial cells. Therefore, we believe that lonicerin has a good protective effect on diabetic-induced endothelial injury and can be applied to the preparation of drugs for treating vascular injury.

[0006] In order to achieve the above-mentioned invention object, the present invention provides the following technical solutions:

[0007] The present invention provides the application of lonicerin in the preparation of drugs for treating vascular injury.

[0008] Preferably, the vascular injury is vascular endothelial injury.

[0009] Preferably, the vascular injury is vascular injury caused by diabetes.

[0010] The present invention also provides a drug for treating vascular injury, and the drug contains lonicerin.

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

[0012] The present invention has found through experimental research that lonicerin has a significant therapeutic effect on treating diabetic-induced vascular injury, and its action pathway is the Ca 2+ pathway. This study shows that lonicerin, as an important active ingredient of Lonicera, is widely distributed, easily available, and low in cost, and is expected to become a new traditional Chinese medicine preparation for treating vascular injury, and may play an important role in the occurrence and development of clinical treatment of vascular injury in the future. Brief Description of the Drawings

[0013] Figure 1 Morphological staining of aortic sections of mice in each group; A. HE staining. B. Masson staining.

[0014] Figure 2 Effects of lonicerin on diabetes-induced vascular calcification; A. ELISA was used to detect the ALP activity in vascular homogenates of each group. B. qPCR was used to detect the levels of BMP2 in the aorta of each group. C. qPCR was used to detect the levels of BMP2 in HAECs of each group. (*p<0.05, **p<0.01, ****p<0.0001, n = 3).

[0015] Figure 3 Effects of lonicerin on diabetes-induced vascular inflammation; A. ELISA was used to detect the content of MCP-1 in the serum of mice in each group. B. qPCR was used to detect the levels of ICAM-1 in the aorta of each group. C. qPCR was used to detect the levels of MCP-1 in HAECs of each group. D. qPCR was used to detect the levels of ICAM-1 in HAECs of each group. (**p<0.01, ***p<0.001, ****p<0.0001, n = 3).

[0016] Figure 4 Effects of lonicerin on diabetes-induced vascular fibrosis; A. Western blot was used to detect the expression of α-SMA in the aorta of mice in each group. B. Western blot was used to detect the expression of Col1al and α-SMA in HAECs of each group. (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, n = 3).

[0017] Figure 5 Effects of lonicerin on diabetes-induced endothelial EndMT; A. Results of immunofluorescence detection of the expression of EndMT markers CD31 and α-SMA in aortic sections of each group. B. Results of immunofluorescence detection of the expression of EndMT markers VE-cadherin and Vimentin in HAECs of each group. (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, n = 3).

[0018] Figure 6 Effects of lonicerin on endothelial function under high glucose stimulation; A. Tube formation assay was used to detect the number and length of tubes formed in each experimental group of HAECs. B. Invasion assay was used to detect the number of invaded cells in each experimental group of HAECs. (**p<0.01, ***p<0.001, ****p<0.0001, n = 4).

[0019] Figure 7 Screening and verification of lonicerin on Ca by whole transcriptome sequencing2+ Effect of the pathway; A. Enrichment pathway map of up TOP 20 in the high glucose group vs. the control group, and enrichment pathway map of down TOP 20 in the high glucose + lonicerin group vs. the high glucose group. B and C. Immunofluorescence detection of the expression level of Ca 2+ in HAECs. (**p<0.01, n = 3). Detailed implementation manners

[0020] Example 1

[0021] 1 Materials and instruments

[0022] Lonicerin (purity 98%) Shanghai Yuanye Bio-Technology Co., Ltd.

[0023] Streptozotocin Shanghai Yuanye Bio-Technology Co., Ltd.

[0024] HE staining kit Wuhan Sevier Bio-Technology Co., Ltd.

[0025] Masson trichrome staining kit Beijing Solarbio Science & Technology Co., Ltd.

[0026] Matrigel matrix glue Corning, USA

[0027] Crystal violet staining solution Beyotime Biotechnology

[0028] TRIS Beijing Solarbio Science & Technology Co., Ltd.

[0029] ECM (Endothelial Cell Medium) Sciencell, USA

[0030] Individual PCR Tubes 8-tube strip, clear Bio Rad, USA

[0031] 0.2ml Flat PCRTube 8-Cap Strips Bio Rad, USA

[0032] Multiplate TM 96-Well PCR Plates Bio Rad, USA

[0033] 'C'PCR Plate Sealing Film Bio Rad, USA

[0034] Bovine Plasma Fibronectin (BPF) Sciencell, USA

[0035] HiScript III RT SuperMix for qPCR, Nanjing Novoprotein Scientific Inc., Ltd.

[0036] ChamQ SYBR qPCR MasterMix, Nanjing Novoprotein Scientific Inc., Ltd.

[0037] TRIZOL, Nanjing Novoprotein Scientific Inc., Ltd.

[0038] Triton X-100, biofroxx Germany

[0039] 4% Paraformaldehyde Solution, Wuhan Sevier Biotechnology Co., Ltd.

[0040] Fetal Bovine Serum, Hyclone USA

[0041] Pre-stained Marker 10 - 170kD, Thermo USA

[0042] 1*PBS Skim Milk Powder, Sangon Biotech (Shanghai) Co., Ltd.

[0043] Transwell 8μm Membrane Pore Size Chamber, Corning USA

[0044] SDS-PAGE Protein Loading Buffer (5X), Beyotime Biotechnology

[0045] BCA Protein Assay Kit (Enhanced), Beyotime Biotechnology

[0046] SDS-PAGE Gel Rapid Preparation Kit, Beyotime Biotechnology

[0047] BeyoECL Star (Ultra-sensitive ECL Chemiluminescence Kit), Beyotime Biotechnology

[0048] PVDF Membrane, Immobilon-P USA

[0049] Tubulin Antibody (Mouse Monoclonal Antibody), Beyotime Biotechnology

[0050] Horseradish Peroxidase (HRP)-conjugated Goat Anti-Rabbit IgG (H+L), Beyotime Institute of Biotechnology; Horseradish Peroxidase (HRP)-conjugated Goat Anti-Mouse IgG (H+L), Beyotime Institute of Biotechnology; Alexa Fluor 555-conjugated Donkey Anti-Rabbit IgG (H+L), Beyotime Institute of Biotechnology; Alexa Fluor 555-conjugated Donkey Anti-Mouse IgG (H+L), Beyotime Institute of Biotechnology; Alexa Fluor 488-conjugated Goat Anti-Rabbit IgG (H+L), Beyotime Institute of Biotechnology; Alexa Fluor 488-conjugated Goat Anti-Mouse IgG (H+L), Beyotime Institute of Biotechnology; Goat Serum, Beyotime Institute of Biotechnology

[0051] DAPI Staining Solution, Beyotime Institute of Biotechnology

[0052] Glycine, Beijing Solarbio Science & Technology Co., Ltd.

[0053] SDS, Beijing Solarbio Science & Technology Co., Ltd.

[0054] Tween-20, Beijing Solarbio Science & Technology Co., Ltd.

[0055] Phosphatase Inhibitor Cocktail, Beyotime Institute of Biotechnology

[0056] RIPA Lysis Buffer (Strong), Beyotime Institute of Biotechnology

[0057] Rhod2, AM, Cell-Permeant Calcium Fluorescent Probe, Nanjing Vazyme Biotech Co., Ltd.; CD31 Rabbit Monoclonal Antibody, Abcam (USA)

[0058] Vimentin Rabbit Monoclonal Antibody, Abcam (USA); VE-cadherin Mouse Monoclonal Antibody, Proteintech (China)

[0059] FSP1 Rabbit Monoclonal Antibody, Abcam (USA)

[0060] Col1a1 Mouse Monoclonal Antibody, Proteintech (China)

[0061] α-SMA Mouse Monoclonal Antibody, Abcam (USA)

[0062] 2 Primer Sequences

[0063] Table 1 Primer Sequences

[0064]

[0065] As shown in SEQ ID NO: 1 - 12

[0066] 3 Experimental Methods

[0067] 3.1 HE Staining

[0068] After 40 minutes of rewarming treatment of the frozen cross-sectional aortic slices, fix them with methanol, rinse with tap water, stain with hematoxylin in a water bath heated to 50 °C, rinse with tap water, differentiate with a differentiating solution, rinse with tap water, blue in warm water, rinse with 80% ethanol, stain with eosin staining solution, rinse with 80% ethanol, rinse with 95% ethanol, rinse with absolute ethanol, clear with xylene, and air dry. Observe under a microscope after mounting the slides.

[0069] 3.2 Masson staining

[0070] After 40 minutes of rewarming treatment of the frozen cross-sectional aortic slices, fix them with 10% neutral formaldehyde and Bouin's fixative overnight, rinse with distilled water, stain with Masson's mixture, rinse with distilled water, differentiate with an acidic ethanol differentiating solution, rinse with distilled water, blue with a blueing solution, rinse with distilled water, stain with ponceau fuchsin staining solution, wash with a weak acid working solution, differentiate with phosphomolybdic acid, wash with a weak acid working solution, stain with aniline blue, wash with a weak acid working solution, rinse with 95% ethanol, rinse with absolute ethanol, clear with xylene, and air dry. Observe under a microscope after mounting the slides.

[0071] 3.3 Alkaline phosphatase (ALP) activity detection experiment

[0072] Weigh about 15 mg of aortic tissue, place it in a sterilized centrifuge tube, add an appropriate volume of lysis buffer, grind the tissue to make a homogenate, and sequentially add the homogenate sample, buffer, substrate solution, and chromogenic agent to configure a reaction system according to the instructions. Gently shake the well plate to mix evenly. Measure the absorbance value of each well at a wavelength of 520 nm using an enzyme-linked immunosorbent assay (ELISA) reader.

[0073] 3.4 MCP-1 serum content detection experiment (ELISA method)

[0074] Add 100 μL of serum sample to the corresponding wells, incubate at room temperature in the dark for 120 min, wash the plate, then add 100 μL of biotinylated antibody per well, incubate at room temperature in the dark for 60 min, wash and dry, add horseradish peroxidase-labeled antibody, then add 100 μL of chromogenic agent TMB solution per well, incubate at room temperature in the dark for 15 min. Add the stop solution, mix well and immediately measure the A 450 value.

[0075] 3.5 Western blot

[0076] Tissues or cells are lysed in RIPA buffer containing protease inhibitors and phosphatase inhibitors. After quantification, load 80 μg of protein sample onto a 10% or 12% SDS-PAGE gel, electrophorese, and then transfer it to a nitrocellulose membrane. Block it with 5% non-fat milk dissolved in PBS for 2 h, then incubate with the primary antibody overnight at 4 °C. Tubulin is used as an internal reference. The next day, after incubating with the corresponding secondary antibody of the same species, develop the film with a developer.

[0077] 3.6 Immunofluorescence staining

[0078] Tissue immunofluorescence: Take out the frozen sections and perform rewarming treatment for 45 min. After washing three times with PBS, permeabilize with permeabilization solution for 1.5 h, wash with PBS, block with blocking solution for 1.5 h, and wash to remove the reaction solution. Add the primary antibody, incubate overnight at 4 °C and then rewarm for 30 min, wash with PBS, add the corresponding species fluorescent secondary antibody and incubate for 1.5 h, wash, slightly dry and then mount with an anti-fluorescence quenching mounting medium containing DAPI, and observe with a confocal microscope.

[0079] Cell immunofluorescence: Seed cells in a 24-well plate containing coverslips and culture. When the growth density reaches about 60%-75%, add drugs to treat the cells. Discard the liquid in the wells, fix with 4% paraformaldehyde for 15 min, permeabilize with 0.1% Triton X-100 at room temperature for 20 min, block with goat serum at room temperature for 1 h, and add the primary antibody and incubate overnight. The next day, add the fluorescent secondary antibody and incubate at room temperature in the dark for 1 h, and stain the cell nuclei with an anti-fluorescence quencher containing DAPI. Collect images with a laser confocal microscope.

[0080] 3.7 qPCR

[0081] Using the reverse transcription product cDNA as a template, add 5.0 μL of TB Green Mix, 2.0 μL of cDNA, 2.0 μL of ddH 2 O, 0.5 μL of PCR Forward Primer, and 0.5 μL of PCR Reverse Primer into an EP tube sterilized by high-pressure steam. GAPDH is used as an internal reference, and BMP2, ICAM-1, and MCP-1 are target genes. The primer sequences of the target genes are shown in Table 1. First, perform pre-denaturation to open the DNA double strands. Subsequently, denature at 95 °C for 15 s, anneal at 50 °C - 65 °C for 1 min, and extend at 72 °C for 10 s. This process is cycled 35 times to detect the relative expression levels of specific genes.

[0082] 3.8 Tube formation assay

[0083] Add Matrigel to a 48-well plate at a volume of 50 μL per well and polymerize at 37 °C for 30 min. Then, place 100 μL of cell suspension on the Matrigel and further culture at 37 °C for 3 - 6 h. Images of each sample are captured by a fluorescence microscopic cell imager, and the angiogenesis ability of cells is quantified using ImageJ software by the number of branch points.

[0084] 3.9 Transwell assay

[0085] The Transwell chamber is divided into an upper chamber and a lower chamber. HAECs are placed in the upper chamber for culture, and the lower chamber contains an extracellular matrix with 10% serum to stimulate cell migration. After 24 hours of free migration, the upper chamber is fixed with 4% paraformaldehyde, and the non-invasive cells on the upper surface of the chamber are gently wiped with a cotton swab, washed 3 times with PBS, incubated with crystal violet staining solution for 10 minutes, and then imaged and photographed with an optical microscope.

[0086] 3.10 Animal culture and model establishment

[0087] (1) The experiment was divided into a normal group, a model (diabetes) group, a model + low-dose loganin group (3 mg / kg / d), a model + medium-dose loganin group (10 mg / kg / d), and a model + high-dose loganin group (30 mg / kg / d). The mice were randomly and evenly assigned to each group.

[0088] (2) Establishment of diabetes model: Except for the normal group, the model group, the model + low-dose loganin group, the model + medium-dose loganin group, and the model + high-dose loganin group were all intraperitoneally injected with 80 mg / kg streptozotocin (STZ) solution for 7 days (mice with fasting blood glucose higher than 11.1 mmol / L were considered to have successfully established the model and were used for subsequent experiments). The normal group mice were intraperitoneally injected with an equal volume of citrate buffer (pH 4.5).

[0089] (3) The normal group and the model group were intragastrically administered 0.5% sodium carboxymethylcellulose (10 mL / kg / d), and the loganin (low, medium, and high-dose groups) was intragastrically administered 3 mg / kg / d, 10 mg / kg / d, and 30 mg / kg / d respectively. All drugs were dissolved in an equal volume of 0.5% sodium carboxymethylcellulose and administered for 10 days.

[0090] 3.11 Cell culture and model establishment

[0091] (1) The experiment was divided into a normal group, a high-glucose group, a high-glucose + low-dose loganin group (3 μM), a high-glucose + medium-dose loganin group (10 μM), and a high-glucose + high-dose loganin group (30 μM).

[0092] (2) When the cell density reached 80% - 90%, the cells were digested with trypsin, and the resuspended cells were respectively inoculated into cell culture plates. When the cell density reached 70% - 80%, drugs were added. The normal group cells were cultured in ordinary ECM medium, the high-glucose group cells were cultured in 30 mM high-glucose medium, the high-glucose + low-dose loganin group cells were cultured in 30 mM high-glucose + 3 μM loganin medium, the high-glucose + medium-dose loganin group cells were cultured in 30 mM high-glucose + 10 μM loganin medium, and the high-glucose + high-dose loganin group cells were cultured in 30 mM high-glucose + 30 μM loganin medium. After adding the drugs, the cells were cultured for 24 hours.

[0093] 3.13 Data statistics

[0094] The experimental result pictures were statistically analyzed using Image J software. The experimental data were all expressed as means ± standard error of the mean (SEM), and analyzed and compared using One-Way ANOVA. Statistical analysis was performed using GraphPad Prism 6.0 software, with p < 0.05 as the standard for significant difference.

[0095] 4 Experimental results

[0096] 4.1 Lonicerin inhibits diabetes-induced vascular intimal injury and collagen deposition

[0097] The aortic intima of mice in the normal group was smooth, the endothelial cells were flat and intact, and there were no obvious pathological changes; compared with the normal group, the integrity and continuity of the aortic intima in the diabetic model group were damaged, and the intimal collagen content increased. Compared with the diabetic model group, after treatment with lonicerin (at a dose of 30 mg / kg), these symptoms could be significantly improved, manifested as relatively neat arrangement of vascular endothelial cells and less secreted intimal collagen ( Figure 1 ).

[0098] 4.2 Lonicerin inhibits diabetes-induced vascular calcification

[0099] Alkaline phosphatase (ALP) is a key enzyme in the early stage of vascular calcification. First, an Elisa experiment was used to detect the activity of ALP in the aortic tissues of each experimental group. The results showed that the activity of ALP in the vascular tissue homogenate of the diabetic model group was significantly increased, and after treatment with lonicerin on the basis of the diabetic model, the activity of ALP in the vascular tissue homogenate was significantly decreased ( Figure 2 A). BMP2 is another representative marker of vascular calcification. The expression of bone morphogenetic protein 2 (BMP2) in the vascular tissues of each experimental group was detected by qPCR. The results showed that lonicerin could significantly down-regulate the increase of BMP2 in the blood vessels of diabetic mice ( Figure 2 B). It indicates that lonicerin can reduce vascular calcification, and the treatment effect of lonicerin is concentration-dependent. Then, to further verify the effect of lonicerin on vascular calcification at the in vitro level, we detected the changes in the expression of the calcification factor BMP2 in each experimental group of HAECs. The results showed that compared with the normal group, high glucose increased the expression of BMP2 in HAECs, while after adding lonicerin treatment on the basis of the high glucose model, the expression level of BMP2 in HAECs was down-regulated ( Figure 2 C).

[0100] 4.3 Lonicerin inhibits diabetes-induced vascular inflammation

[0101] To verify the effect of lonicerin on inflammation after endothelial injury, the expression levels of the endothelial injury-related inflammatory markers intercellular cell adhesion molecule-1 (ICAM-1) and monocyte chemotactic protein 1 (MCP-1) in the samples of each experimental group were detected by ELISA or qPCR in vitro and in vivo. First, the in vivo detection results showed that compared with the normal group, the diabetic model group significantly induced the expression of the inflammatory factor MCP-1 in the serum, while the content level of MCP-1 decreased after co-treatment with lonicerin and showed a concentration gradient-dependent trend ( Figure 3 A). Meanwhile, the aortic qPCR results showed that compared with the normal group, the expression level of ICAM-1 in the aortic samples of the diabetic model group of mice increased, and compared with the model group, the expression level of ICAM-1 decreased after treatment with lonicerin ( Figure 3 B). In addition, the in vitro results showed that high-glucose medium induced an increase in the expression of the inflammatory factors ICAM-1 and MCP-1 in HAECs, and after treatment with lonicerin, the increased expression levels of ICAM-1 and MCP-1 induced by high glucose were significantly reduced ( Figure 3 C, D). These results indicate that lonicerin has a protective effect on vascular inflammatory injury in diabetic mice.

[0102] 4.4 Lonicerin inhibits diabetes-induced vascular fibrosis

[0103] To verify the effect of lonicerin on vascular fibrosis, Western blot was used to analyze the expression of the fibrosis markers α-SMA and collagen, type I, alpha 1 (Col1a1) in each experimental group. The results showed that in the in vivo experiment, the diabetic model induced vascular fibrosis and promoted the up-regulation of the expression of the vascular fibrosis marker α-SMA. After treatment with lonicerin, the expression of α-SMA in vascular tissues could be down-regulated, alleviating vascular fibrosis ( Figure 4 A). The in vitro experimental results were consistent with the in vivo results. The high-glucose model could induce an increase in the expression of α-SMA and Col1a1 in HAECs. After co-culture with lonicerin added, the expression of α-SMA and Col1a1 in HAECs was down-regulated ( Figure 4 B).

[0104] 4.5 Lonicerin inhibits diabetes-induced endothelial to mesenchymal transition (EndMT)

[0105] Previous studies have shown that EndMT plays an important role in diabetes-induced vascular injury. To explore the effect of lonicerin on EndMT in vascular endothelial cells under diabetic conditions, immunofluorescence double staining of CD31 and α-SMA was performed on vascular cross-section slices at the in vivo level, and immunofluorescence double staining of VE-cadherin and Vimentin was performed on HAECs in vitro. The fluorescence staining results of aortic slices showed that compared with the normal group, the content of CD31 decreased and the expression of α-SMA increased in the diabetic model group, and the two proteins were co-localized in the aortic intima, indicating the occurrence of EndMT in the diabetic model group. After treating the blood vessels with EndMT with lonicerin, the expression of CD31 increased and the expression of α-SMA decreased in the intimal tissue of the blood vessels, and the process of EndMT was reversed( Figure 5 A). In in vitro experiments, compared with the normal group, high glucose induced an increase in the expression of Vimentin and inhibited the expression of VE-cadherin, while co-culture with lonicerin promoted the expression of VE-cadherin, down-regulated the expression of Vimentin, and inhibited the process of EndMT( Figure 5 B).

[0106] 4.6 Lonicerin protects vascular endothelial function from high glucose injury

[0107] Tube formation ability is one of the specific functions of endothelial cells, and invasion ability is one of the functions of mesenchymal cells. Tube formation assay and invasion assay were used to detect the tube formation and invasion ability of HAECs. The results showed that compared with the normal group, HAECs in the high glucose group had poor tube formation ability (manifested as lower tube formation number and tube formation length) and enhanced invasion ability. After administration of lonicerin on this basis, the tube formation ability of HAECs was restored (the number and length of tube formation increased), and the invasion ability was inhibited (the number of cells penetrating the basement membrane decreased), indicating that lonicerin treatment inhibited the process of EndMT and restored endothelial function( Figure 6 ). 4.7 Lonicerin protects blood vessels endothelium by regulating Ca 2+ pathway

[0108] To explore the mechanism of action of lonicerin in alleviating vascular endothelial injury, HAECs were divided into a normal group, a high glucose group, and a high glucose + lonicerin group, and the differential gene expression in HAECs was detected by whole transcriptome sequencing. The KEGG results showed that the Ca 2+ signal pathway (calcium signal passway) was significantly enriched in the top 20 pathways of high glucose group VS control group and the top 20 pathways of high glucose + lonicerin group VS high glucose group. It is speculated that lonicerin may play a role by inhibiting the Ca 2+ signal pathway. After verification, under high glucose conditions, Ca 2+The concentration increased, and after administration of lonicerin, the Ca 2+ concentration decreased ( Figure 7 ). This result indicates that lonicerin may protect vascular endothelium by regulating the Ca 2+ pathway.

[0109] In this study, we first found that lonicerin could significantly inhibit vascular damage caused by diabetes through experimental methods such as HE staining and Masson staining. By means of Elisa, Western blot, qPCR, etc., it was found that lonicerin could significantly inhibit the up-regulation of calcification factors, inflammatory factors, and fibrosis factors in vascular tissues induced by diabetes. In addition, through methods such as immunofluorescence experiments, tube formation experiments, and invasion experiments, it was determined that lonicerin also has an inhibitory effect on high glucose-induced EndMT and promotes the recovery of related endothelial functions of HAECs. Secondly, through whole transcriptome sequencing, it was first discovered and determined that the biological effect of lonicerin in protecting vascular endothelium is related to the Ca 2+ signaling pathway.

[0110] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. Application of honeysuckle in the preparation of drugs for treating vascular damage.

2. The use according to claim 1, characterized in that: The vascular damage is vascular endothelial damage.

3. The use according to claim 1, characterized in that: The vascular damage is vascular damage caused by diabetes.

4. A drug for treating vascular damage, characterized in that: The medicine contains honeysuckle glycoside.

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