Application of octyl 4-itaconate in relieving and treating peripheral vascular diseases

Abstract

The invention discloses application of octyl 4-itaconate in preparation of a medicine for promoting proliferation, migration or scratch healing of vascular endothelial cells, and belongs to the technical field of medicines for vascular diseases. The medicine is 4-octyl itaconate, the administration dosage of the medicine is 25-30 mg / kg of body weight, and the concentration of the 4-octyl itaconate in the medicine is 5-8 mg / mL. The octyl 4-itaconate can promote proliferation, migration or scratch healing of vascular endothelial cells, so that blood flow of lower limb blood vessels after lower limb ischemia reperfusion is remarkably recovered, lesions of lower limb muscular tissues are relieved, new blood capillaries are promoted, and the curative effect is improved. Therefore, the compound has a wide clinical application prospect in preparation of drugs for promoting proliferation, migration or scratch healing of vascular endothelial cells.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology for vascular diseases, and particularly relates to the application of octyl 4-itaconate in the relief and treatment of peripheral vascular diseases. Background Technology

[0002] In recent years, the incidence of peripheral vascular disease has reached 3%-10%, with approximately 5,000-10,000 people per million population suffering from lower extremity vascular disease each year. Lower extremity ischemic disease is a common peripheral vascular disease, caused by various factors (such as diabetes, hyperlipidemia, hypertension, or local and systemic inflammatory responses resulting from vascular surgery that blocks blood flow) leading to stenosis or occlusion of lower extremity arteries, resulting in insufficient tissue blood perfusion. This leads to ischemia, hypoxia, tissue cell degeneration, and necrosis in the distal tissues of the arteries. Clinically, it mainly manifests as intermittent claudication, cold and numb limbs, and rest pain. In severe cases, ulcers and gangrene may occur, even ultimately leading to amputation and death, seriously threatening human health.

[0003] Angiogenesis is divided into angiogenesis, vascularization, and arterial formation. Pathogenesis studies show that the main process of angiogenesis involves the dissolution of the junctions between endothelial cells and the basement membrane, endothelial cell migration, adhesion, and reconnection, ultimately forming new capillary lumens. When ischemia occurs, hypoxia and inflammation are the main stimulating factors for angiogenesis. The ischemic site can rapidly compensate by generating new blood vessels, maintaining blood supply to the ischemic area by establishing effective collateral circulation, thus reducing or preventing ischemia and necrosis. Therefore, the speed and quantity of angiogenesis are two important indicators directly related to the prognosis of ischemic diseases. Enabling rapid compensatory angiogenesis in ischemic sites is considered a crucial measure for treating ischemic diseases such as peripheral vascular disease, and it is currently a research hotspot and a major problem urgently needing to be solved in this field.

[0004] Itaconic acid (4-Octyl Itaconate, an unsaturated dicarboxylic acid) is an important intermediate metabolite in the tricarboxylic acid cycle, synthesized by the decarboxylation of cis-aconate catalyzed by cis-aconate decarboxylase encoded by the immunoreactivity gene-1 (IRG-1) in the mitochondrial matrix. In recent years, itaconic acid has attracted considerable attention from researchers due to its anti-inflammatory and immunomodulatory effects. Studies have found that under inflammatory response conditions in macrophages and bone marrow cells, such as stimulation by LPS, itaconic acid accumulates and increases intracellularly as an important immunomodulatory metabolite. A series of studies have shown that itaconic acid has significant anti-inflammatory effects and is central to the connection between immunity, metabolism, and inflammation (Bambouskova et al. Nature. 2018, 556(7702):501-4). However, there are currently no reports on the related functions of octyl 4-itaconate in vascular endothelial cells and peripheral vascular diseases. Summary of the Invention

[0005] To address the shortcomings of the existing technology, this invention provides an application of octyl 4-itaconate in the preparation of drugs that promote the proliferation, migration, or scratch healing of vascular endothelial cells, thus providing a new use for octyl 4-itaconate, specifically achieved through the following technology: In a first aspect, the present invention provides an application of octyl 4-itaconate, (1) in the preparation of a medicament for promoting the proliferation, migration or scratch healing of vascular endothelial cells; and (2) in the promotion of the proliferation, migration or scratch healing of vascular endothelial cells not for the purpose of disease treatment or diagnosis.

[0006] It should be noted that the "promotion of vascular endothelial cell proliferation, migration or scratch healing not for the purpose of disease treatment and diagnosis" mentioned in the above applications generally refers to pathological scientific research, drug screening and efficacy research in the laboratory, and does not refer to clinical diagnosis and treatment in humans or animals.

[0007] Furthermore, the 4-itaconic octyl ester is used to prepare a drug for relieving / treating peripheral vascular disease.

[0008] Furthermore, the dosage of the drug is 25-50 mg / kg based on the body weight of the organism.

[0009] Furthermore, the concentration of octyl 4-itaconate in the drug is 5-8 mg / mL.

[0010] Furthermore, the aforementioned peripheral vascular diseases include peripheral vascular injury caused by lower limb ischemia-reperfusion.

[0011] Peripheral vascular injury is generally caused by poor blood circulation and tissue lesions.

[0012] Furthermore, the drug is an oral or injectable preparation, meaning it is administered orally or by injection.

[0013] This invention utilizes a mouse model of lower limb ischemia-reperfusion to verify the efficacy of octyl 4-itaconate. Results showed that after lower limb ischemia-reperfusion in mice, blood flow in the lower limb vessels decreased significantly, and lesions occurred in the lower limb muscle tissue. Treatment with octyl 4-itaconate significantly restored blood flow in the lower limb vessels, alleviated the lesions in the lower limb muscle tissue, and promoted the regeneration of lower limb capillaries. To verify the mechanism of these effects, the inventors conducted a series of in vitro cell experiments. The results showed that octyl 4-itaconate can promote the proliferation, migration, and scratch healing of vascular endothelial cells. These results provide a theoretical basis for the use of octyl 4-itaconate in improving and repairing peripheral vascular injury.

[0014] Compared with the prior art, the advantages of the present invention are: 1,4-Octanol ester can promote endothelial cell proliferation, migration, and scratch healing, thereby facilitating the recovery and reconstruction of damaged blood vessels and providing a new option for the preparation of drugs that promote vascular endothelial cell proliferation, migration, or scratch healing.

[0015] 2,4-Octanol can restore blood flow in the lower limb vessels after ischemia-reperfusion and significantly reduce lesions in the lower limb muscle tissue, thus showing broad clinical application prospects in the preparation of drugs to relieve or treat peripheral vascular injury caused by lower limb ischemia-reperfusion. Attached Figure Description

[0016] Figure 1 The images shown are representative laser Doppler images of the mouse legs before and after surgery for lower limb ischemia (left ischemic, right non-ischemic) at 0d, 3d, 7d and 14d after surgery as described in Example 1. Sham is the sham surgery group, HLI is the lower limb ischemia-reperfusion group, Veh is the control group, and 4-OI is the 4-OI treatment group. Figure 2 The figure shows the results of blood flow recovery after ischemia in mice as described in Example 1. In the figure, Veh is the control group, 4-OI is the 4-OI treatment group, HLI+Veh is the lower limb ischemia-reperfusion surgery model group, and HLI+4-OI is the lower limb ischemia-reperfusion surgery plus 4-OI treatment group. (HLI+Veh vs Veh, *p<0.05, **p<0.01; HLI+4-OI vs HLI+Veh, #p<0.05, ##p<0.01, n=6). Figure 3 This is a diagram showing the morphological results of the mouse lower limb muscle tissue as described in Example 2; Figure 4 The image shows the CD31 immunofluorescence detection results (50 μm, n=6) described in Example 3. Figure 5 The effect of different concentrations of 4-OI on the proliferation of HUVECs as described in Exercise 4 is shown in the figure (*p<0.05, **p<0.01, n=6). Figure 6 The figure shows the effect of 4-OI stimulation for different durations on HUVECs cell proliferation as described in Example 4 (*p<0.05, **p<0.01, n=6). Figure 7 This is a graph showing the effect of 4-OI stimulation on scratch healing of HUVECs cells as described in Example 5; Figure 8 The graph shows the quantitative results of 4-OI stimulation on scratch healing of HUVECs cells as described in Example 5 (*p<0.05, **p<0.01, ****p<0.0001, n=6). Figure 9This is a graph showing the effect of 4-OI stimulation on HUVECs cell migration as described in Example 6; Figure 10 The graph shows the quantitative results of the 4-OI stimulation on the migration rate of HUVECs cells described in Example 6 (*p<0.05, **p<0.01, ****p<0.0001, n=6). Figure 11 The figure shows the effect of 4-OI on the formation of cell lumen in HUVECs as described in Example 7. Detailed Implementation

[0017] The technical solution of the present invention will be clearly and completely described below. 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 skilled in the art without creative effort are within the scope of protection of the present invention.

[0018] Example 1: Effect of octyl 4-itaconate (4-OI) on blood flow after hind limb ischemia (HLI) reperfusion in mice.

[0019] The experimental animals included 30 male SPF-grade C57 mice (Hubei Provincial Center for Disease Control and Prevention), aged 6-8 weeks and weighing 18-20 g. During the rearing period, the room temperature was maintained at approximately 22℃, the relative humidity at approximately 50%, with 12-12 hours of light, free access to water and food. After randomization, the mice were divided into four cages and acclimatized in the laboratory for one week. They were then randomly assigned to four groups: sham surgery group, model group (HLI), model plus drug group (HLI+4-OI), and white drug group (sham+4-OI). Surgical modeling and group feeding were then performed.

[0020] 1. Establishing a lower limb ischemia model and administering medication.

[0021] (1) Modeling Anesthetized the mice with an intraperitoneal injection of 10% chloral hydrate solution at a dose of 0.06 mL / 10 g. After complete anesthesia and weakened paw reflexes, the lower abdomen and bilateral hind limbs were treated with depilatory cream. The mice were fixed in a supine position on the operating board and placed under a stereomicroscope. The left hind limb was used for the procedure. After disinfection with povidone-iodine, a 1 cm incision was made in the groin along the abdominal cavity using ophthalmic scissors. Subcutaneous and adipose tissues were bluntly dissected transversely. Under stereomicroscope, the femoral artery, femoral vein, and femoral nerve were separated using fine forceps, and the femoral artery and its main branches were isolated. After separating the femoral artery, a 7-0 suture was passed below the proximal end of the femoral artery and occluded with a double knot. At the distal end of the femoral artery, a 7-0 suture was passed below the distal femoral artery and ligated. The femoral artery was transected at the midpoint between the ligations at both ends. After disinfection with povidone-iodine, the skin was sutured with 4-0 sutures.

[0022] (2) Administration Normal feed was provided; fasting and water restriction were not required. Before modeling, the HLI+4-OI group and the 4-OI group were pretreated for three days by intraperitoneal injection of 5 mg / mL 4-OI at a dose of 25 mg / kg. After modeling, the same dose and concentration of 4-OI were injected intraperitoneally daily for two weeks. The HLI group and the sham group received a control control treatment of intraperitoneal injection of 95% β-cyclodextrin + 5% DMSO solvent for two weeks.

[0023] 2. Laser Doppler flowmeter detects blood flow.

[0024] (1) Animal preparation Before the procedure, mice were anesthetized intraperitoneally with a 10% chloral hydrate solution. After complete anesthesia, the skin on the abdomen and below of the mice was shaved. The shaved mice were placed supine on a black background board with their limbs extended and properly secured.

[0025] (2) Select the measurement area The laser speckle blood flow imaging system was activated, and the laser emission point height was fixed. Using the PIMsoft imaging system, the measurement area of ​​the lower limbs was selected, covering the mouse's lower limbs and feet. The selected measurement area was the same on both lower limbs. Blood flow in the lower limbs was measured, and the quantitative blood flow result was the laser speckle scan value within the selected area. The blood flow data of the lower limbs was expressed as the ratio of the values ​​on the operated side to the unoperated side to reduce the influence of environmental changes at different measurement times on the blood flow values.

[0026] (3) Interpretation of blood flow On the day of mouse modeling, mice were given intraperitoneal anesthesia and fixed in position before surgery. Lower limb blood flow was measured. Immediately after modeling, lower limb blood flow was measured to verify the model and record the lower limb ischemia status on postoperative day 0. The unoperated lower limb served as a self-control. Lower limb blood flow was measured and recorded on postoperative days 3, 7, and 14 after anesthesia. All results are expressed as the ratio of laser speckle blood flow reading on the operated side to the blood flow reading on the unaffected side.

[0027] (4) Statistical analysis (quantitative) of blood flow data in the lower limbs of mice was performed using Graphpad Prism 8. The results are shown in Table 1 below.

[0028] Table 1. Blood flow in mice after hind limb ischemia.

[0029] Representative laser Doppler images of mouse legs before and at 0, 3, 7, and 14 days after surgery for hind limb ischemia are shown in the experimental results. Figure 1As shown in the figure, it can be seen that whether or not 4-OI intervention was used in the sham surgery group had no significant effect on blood flow in the lower limbs of mice, while 4-OI intervention significantly improved blood flow recovery after ischemia in the lower limbs of mice.

[0030] The ratio of perfusion in ischemic to non-ischemic hind limbs in mice is used to quantify the recovery of blood flow after hind limb ischemia. Figure 2 As shown, according to Table 1 above and Figure 2 Data showed that intervention with 4-OI significantly improved blood flow to the lower limbs starting on day 7 post-surgery.

[0031] Example 2: Effects of 4-OI on muscle tissue cell morphology after hind limb ischemia (HLI) reperfusion in mice

[0032] Based on Example 1, mouse hind limb muscle tissue was taken and subjected to HE staining analysis. The main steps were as follows: paraffin sections of mouse hind limb muscle were sequentially immersed in xylene twice for 20 min each time, anhydrous ethanol twice for 5 min each time, washed with 75% alcohol for 5 min, rinsed with tap water, and stained with hematoxylin for 5 min. Then, the paraffin sections of mouse hind limb muscle were sequentially rinsed with tap water, differentiated with differentiation solution, washed with tap water, soaked in blue solution, rinsed with tap water, dehydrated in 85% and 95% graded alcohol for 5 min each time, stained with eosin for 5 min, stained with anhydrous ethanol three times for 5 min each time, and xylene twice for 5 min each time to make the sections transparent. Finally, the sections were mounted with neutral resin and examined under a microscope. Images were acquired and analyzed.

[0033] The results are as follows Figure 3 As a result, compared with the sham-operated group, the sarcomere arrangement of the hind limb muscle tissue of mice in the lower limb ischemia group was disordered and there was a large amount of inflammatory cell infiltration. Compared with the lower limb ischemia group, after intervention with 4-OI, the sarcomere arrangement of the hind limb muscle tissue of mice was significantly improved and the inflammatory cell infiltration was significantly reduced. This result shows that 4-OI can significantly improve the lesions of the lower limb muscle tissue caused by lower limb ischemia in mice.

[0034] Example 3: Effects of 4-OI on angiogenesis in muscle tissue after hind limb ischemia-reperfusion in mice 1. Dewaxing paraffin sections to water The slices were placed in xylene I (Sinopharm Chemical Reagent Co., Ltd.) for 5 min, xylene II for 15 min, anhydrous ethanol I (Sinopharm Chemical Reagent Co., Ltd.) for 5 min, anhydrous ethanol II for 5 min, 85% ethanol for 5 min, 75% ethanol for 5 min, and then washed with distilled water.

[0035] 2. Antigen retrieval Tissue slides were placed in a retrieval box filled with EDTA antigen retrieval buffer (pH=8.0) (Wuhan Baiqiandu Biotechnology) and microwaved for antigen retrieval. Microwave on medium heat for 8 minutes, turn off for 8 minutes, then microwave on medium-low for 7 minutes. During this process, excessive evaporation of the buffer should be prevented; the slides should not be allowed to dry. After natural cooling, slides (Jiangsu Shitai Experimental Equipment Co., Ltd.) were placed in PBS (pH=7.4) and washed three times on a decolorizing shaker (Beijing Liuyi Instrument Factory), 5 minutes each time.

[0036] 3. Quenching of spontaneous fluorescence by drawing circles After slightly drying the sections, draw circles around the tissue with a histochemical pen (to prevent antibody migration), add autofluorescence quencher (Wuhan Baiqiandu Biotechnology) inside the circles for 5 minutes, and rinse with running water for 10 minutes.

[0037] 4. Serum blocking Add BSA dropwise into the circle and incubate for 30 minutes.

[0038] 5. Add primary antibody Gently shake off the blocking solution, add the prepared primary antibody in PBS at a specific ratio to the slide, and incubate the slide flat in a humidified chamber at 4°C overnight. (Add a small amount of water to the humidified chamber to prevent antibody evaporation.) 6. Add secondary antibody The slides were placed in PBS (pH=7.4) and washed three times on a decolorizing shaker for 5 minutes each time. After slightly drying the sections, secondary antibody corresponding to the species of the primary antibody was added to the inner circle to cover the tissue, and the tissue was incubated at room temperature in the dark for 50 minutes.

[0039] 7. DAPI counterstaining of cell nuclei The slides were placed in PBS (pH=7.4) and washed three times on a destaining shaker for 5 minutes each time. After slightly drying the sections, DAPI staining solution was added to the inner circle and incubated at room temperature in the dark for 10 minutes.

[0040] 8. Sealing The slides were placed in PBS (pH=7.4) and washed three times on a decolorizing shaker for 5 minutes each time. After slightly drying the sections, they were mounted with anti-fluorescence quenching mounting medium.

[0041] 9. Microscopic examination and photography The sections were observed and images were acquired under a fluorescence microscope.

[0042] Angiogenesis is a crucial repair step following lower limb ischemia; promoting angiogenesis can improve blood perfusion and reduce tissue necrosis. This embodiment uses CD31 immunofluorescence staining to detect capillary density to assess angiogenesis. Results are as follows... Figure 4As shown, compared with the control group, the capillary density in the model group was significantly reduced, while compared with the model group, the capillary density in the 4-OI intervention group was significantly increased. This result indicates that the intervention of 4-OI promotes capillary regeneration after lower limb ischemia-perfusion.

[0043] Example 4: Effect of 4-OI on the proliferation of human umbilical vein endothelial cells (HUVECs) To further verify that 4-OI in Examples 1-3 above can promote lower limb angiogenesis and blood flow restoration and alleviate the underlying causes of muscle tissue lesions, this example verifies the effects of different concentrations and treatment times of 4-OI on the proliferation of human umbilical vein endothelial cells (HUVECs). The implementation steps are as follows: 1. Effects of different concentrations of 4-OI on the proliferation of human umbilical vein endothelial cells (HUVECs) (1) Wash HUVECs cells with PBS (3 generations), digest the treated cells with trypsin (1.5 min), centrifuge at 1000 rpm for 5 min, discard the supernatant, resuspend in 1 mL of ECM (Sciencell) medium, and count the cells; V=N / N 计 =(70×10 4 ) / [(284 / 4)×10 4 =1mL.

[0044] (2) According to 10 per hole 4 The cells were evenly seeded into a plate. 1 mL of cell suspension was mixed with 6 mL of 5% EAM and 100 mL was added to each well. PBS was added around the perimeter of the plate and the plate was incubated overnight at 37°C with 5% CO2.

[0045] (3) Prepare 2% FBS complete culture medium containing the drug. The formula for each group is as follows: 250mM group: 2397.6mL 2% ECM and 2.4mL 250mM 4-OI; 125mM group: 1000mL 2% ECM and 1000mL 250mM 4-OI; 62.5mM group: 700mL 2% ECM and 700mL 125mM 4-OI; DMSO group: 1198.8 mL 2% ECM and 1.2 mL DMSO.

[0046] (4) Set up DMSO, 250mM, 125mM, and 62.5mM 4-OI groups, with 12 replicates in each group. Discard the culture medium in the 96-well plate, add the prepared drug according to the group, and incubate in a 37℃ 5%CO2 incubator for 24h.

[0047] (5) After 24 hours, prepare a 10% CCK-8 solution in the dark, discard the FBS complete medium in the 96-well plate, add 100 mL of 10% CCK-8 solution to each well, and incubate in a 37℃, 5% CO2 incubator for 1 hour.

[0048] One hour later, readings were taken using a ELISA reader (Bio-Tek) at a wavelength of 450 nm. Statistical analysis was performed using a GraphPad Prism 8.

[0049] The results are as follows Figure 5 As shown in Table 2 below, the results show that 4-OI can significantly promote the proliferation of HUVECs, and the proliferation of HUVECs cells increases in a concentration-dependent manner.

[0050] Table 2. Analysis of the effect of 4-OI concentration on HUVEC proliferation

[0051]

[0052] 2. Effects of 4-OI at different treatment times on the proliferation of human umbilical vein endothelial cells (HUVECs) (1) Wash HUVEC cells with PBS (3rd generation), digest the treated cells with trypsin (1 min 30 s), centrifuge at 1000 rpm for 5 min, discard the supernatant, resuspend in 1 mL of ECM medium, and count the cells; V = N / N 计 =(70×10 4 ) / [(284 / 4)×10 4 =1mL.

[0053] (2) According to 10 per hole 4 Spread cells evenly on a plate, add 100 mL to each well, add PBS around the perimeter, and incubate overnight at 37°C with 5% CO2.

[0054] (3) Set up DMSO group and 125mM 4-OI group, with 4 replicates in each group, and process each group according to 0h, 24h, 48h and 72h time.

[0055] (4) Prepare 2% FBS complete culture medium containing the drug at time 0h: 125mM group: 599.4mL 2% ECM and 0.6mL 250mM 4-OI; DMSO group: 599.4 mL 2% ECM and 0.6 mL DMSO.

[0056] (5) Add the drug solution prepared at 0h to the 72h DMSO and 125mM groups respectively, and add 2% ECM to the remaining groups, and incubate in a 37℃, 5% CO2 incubator.

[0057] (6) Prepare 2% FBS complete culture medium containing the drug on the 24th hour: 125mM group: 1198.8mL 2% ECM and 1.2mL 250mM 4-OI; DMSO group: 1198.8 mL 2% ECM and 1.2 mL DMSO.

[0058] (7) The drug solution prepared at 24h was added to the DMSO and 125mM groups at 72h and 48h respectively, and 2% ECM was added to the remaining groups. The mixture was then placed in a 37℃, 5% CO2 incubator for incubation.

[0059] (8) Prepare 2% FBS complete culture medium containing the drug at 48h: 125mM group: 1798.2mL 2% ECM + 1.8mL 250mM 4-OI; DMSO group: 1798.2 mL 2% ECM + 1.8 mL DMSO.

[0060] (9) The drug solution prepared at 48h was added to the DMSO and 125mM groups at 72h, 48h and 24h respectively, and 2% ECM was added to the remaining groups. The mixture was then placed in a 37℃, 5% CO2 incubator for incubation.

[0061] (10) When the time reaches 72h, prepare 10% CCK-8 solution in the dark, discard the culture medium in the 96-well plate, add 100mL of 10% CCK-8 solution to each well, and incubate in a 37℃, 5% CO2 incubator for 0.5h.

[0062] (11) After 0.5 h, the readings were taken using a microplate reader (Bio-Tek) at a wavelength of 450 nm. Statistical analysis of the data was performed using a GraphPad Prism 8.

[0063] The results are as follows Figure 6 As shown in Table 3 below, the results indicate that the proliferation of HUVECs cells increased significantly with the extension of 4-OI treatment time, and the increase in HUVECs cells was time-dependent.

[0064] Table 3. Effects of different 4-OI treatment times on the proliferation of human umbilical vein endothelial cells.

[0065]

[0066] Example 5: Effect of 4-OI on scratch healing of HUVECs cells To further verify that 4-OI can promote lower limb angiogenesis and blood flow recovery and alleviate the underlying causes of muscle tissue lesions in Examples 1-3 above, this example verifies the effect of 4-OI on scratch healing of HUVECs cells. The main steps of the scratch experiment are as follows: 1. Marking the culture plate: First, use a marker pen to draw horizontal lines evenly on the back of the 6-well plate, using a ruler as a guide. Draw one line approximately every 0.5cm, passing through the wells, with 3 lines passing through each well.

[0067] 2. Cell seeding: Wash HUVECs cells with PBS (3rd generation), digest the treated cells with trypsin (1.5 min), centrifuge at 1000 rpm for 5 min, discard the supernatant, add 2 mL of ECM (Sciencell) medium to resuspend, and add approximately the number of cells that can grow to 95% overnight to the wells.

[0068] 3. Cell streaking: On the second day, use the pipette tip to draw horizontal lines perpendicular to the back of the device, with the pipette tip perpendicular and not tilted. Take samples at 0h, and observe the cell migration at specific locations using an inverted microscope and take photos.

[0069] 4. Prepare a complete culture medium containing the drug: 250mM group: 3500mL ECM and 3.5mL 250mM 4-OI; 125mM group: 1500mL ECM and 1500mL 250mM 4-OI; 62.5 mM group: 1000 mL ECM and 1000 mL 125 mM 4-OI; DMSO group: 3500 mL ECM and 3.5 mL DMSO.

[0070] 5. Set up DMSO, 250mM, 125mM, and 62.5mM 4-OI groups. Wash the cells three times with PBS to remove the crossed cells, and add the mixed drugs according to the group.

[0071] 6. Cell culture and observation: Place in a 37℃, 5% CO2 incubator for culture. Take samples at 12 and 24 hours, observe cell migration at specific locations using an inverted microscope and take photos.

[0072] Results Analysis: After opening the images using ImageJ software, 6-8 horizontal lines were randomly drawn, and the mean distance between cells was calculated. Statistical analysis was performed using GraphPad Prism 8.

[0073] Experimental results are as follows Figure 7-8As shown in Table 4 below, compared with 0h, the scratch healing rate of the 4-OI treatment group was significantly improved, and the scratch healing rate also increased with the increase of 4-OI concentration, thus proving that 4-OI has the effect of promoting scratch healing of HUVECs cells.

[0074] Table 4 Statistical analysis of intercellular distances

[0075]

[0076] Example 6: Effects of 4-OI on HUVECs cell migration To further verify that 4-OI can promote lower limb angiogenesis and blood flow recovery and alleviate the underlying causes of muscle tissue lesions in Examples 1-3 above, this example verifies the effect of 4-OI on HUVECs cell migration. The main steps of the scratch assay are as follows: 1. Prepare 20% FBS (Sciencell) complete culture medium containing the drug: 250mM group: 1200mL 20% ECM and 1.2mL 250mM 4-OI; 125mM group: 500mL 20% ECM and 500mL 250mM 4-OI; 62.5mM group: 350mL 20% ECM and 350mL 125mM 4-OI; DMSO group: 600 mL and 0.6 mL DMSO.

[0077] 2. Wet the chamber with PBS (Coring) for 5 min; wash HUVECs cells with PBS (3rd generation), digest the treated cells with trypsin (1.5 min), centrifuge at 1000 rpm for 5 min, discard the supernatant, add 1 mL of serum-free culture medium to resuspend, and count the cells.

[0078] 3. Take out the 24-well plate and set up the DMSO, 250mM, 125mM, and 62.5mM 4-OI (MCE, USA) groups. Add 600mL of 20% ECM medium containing the drug solution to each well. Place the Transwell chamber into the well, and check for air bubbles between the Transwell chamber and the surface of the medium. Observe under a microscope. If air bubbles are present, remove the chamber and place it back in the well.

[0079] 4. Thoroughly pipette the cell suspension until homogeneous. Add 200 μL of serum-free cell suspension to the upper layer of the Transwell chamber (5 × 10⁶ cells per well). 4(1) Avoid cell accumulation in a certain area, which may affect cell penetration efficiency; finally, gently place the 24-well plate into a 37℃, 5% CO2 incubator for 48 hours.

[0080] 5. After 48 hours, discard the culture medium from both chambers, wash both chambers three times with PBS, and gently wipe away the upper layer of cells in the chamber with a cotton swab (gently rotate the cotton swab).

[0081] 6. Cell fixation: Add 600 mL of 4% paraformaldehyde to the lower layer of the Transwell chamber, and fix the upper layer of the chamber in paraformaldehyde (Wuhan Sewell Biotechnology Co., Ltd.) for 20 min.

[0082] 7. Wash the chamber three times with PBS, and gently wipe away the upper layer of cells with a cotton swab (gently rotate the cotton swab).

[0083] 8. Cell staining: After cell fixation, discard paraformaldehyde and add 600 μL of 5% crystal violet solution (Beyotime Biotechnology Co., Ltd.) to each well. Place the Transwell chamber in the crystal violet solution and stain in the dark for 15 min.

[0084] 9. Cell washing: After cell staining, use tweezers to hold the Transwell chamber and gently rinse it in PBS to remove residual dye. Use a cotton swab to gently rotate and wipe away the cells and dye residue on the upper layer of the chamber. Be gentle to avoid damaging the carbonate membrane. Rinse 3-5 times and air dry.

[0085] 10. Cell photography: Take pictures under an inverted microscope (OLYMPUS, Japan), and randomly select 7-8 fields of view for sampling and photography.

[0086] 11. Perform Transwell cell migration counting using ImageJ. The specific steps are as follows: (1) Open the software Image J, create a new file in the file folder, and import the image to be analyzed.

[0087] (2) Click (Image) - (Type) - (8-bit) to convert the image background to black and white.

[0088] (3) Click (Edit) - (Invert) to change the image background to black.

[0089] (4) Click (Image) - (Adjust) - (Threshold), select (B&W), adjust the scroll bar to make the image contain as many cells as possible while removing impurities in the background, and click (Apply) to apply.

[0090] (5) If there are clusters of cells in the image, they need to be segmented. Click (Process) - (Binary) - (Watershed). If there are not many cells, they can be ignored.

[0091] (6) Click (Analyze) - (Analyze Particles) to get the analysis results, where count is the number of transwell-migrated cells.

[0092] (7) The results of the random snapshots were analyzed using ImageJ and compiled into an Excel spreadsheet. Then, GraphpadPrism 8 was used for statistical analysis.

[0093] Experimental results are as follows Figure 9-10 As shown in Table 5 below, intervention with 4-OI significantly promoted the migration of HUVECs cells, and the migration rate gradually increased with increasing 4-OI concentration, reaching its highest level at a concentration of 250 mM, which was approximately 400% higher than the control group. These experimental results demonstrate that 4-OI can significantly promote the migration of HUVECs cells.

[0094] Table 5 Statistical analysis of Transwell-migrated cells

[0095]

[0096] Example 7: Effect of 4-OI on lumen formation in HUVECs cells To further verify the effects of 4-OI on promoting the proliferation, scratch healing, and migration of HUVECs as demonstrated in Examples 4-6 above, this example verified the effect of 4-OI on the lumen formation of HUVECs. The specific steps are as follows: 1. Preparation: The day before the experiment, place the Corning gel in an ice box and let it dissolve overnight at 4°C (while pre-cooling the 200μL and 1000μL pipette tips, EP and 24-well plates at -20°C).

[0097] 2. Plate preparation: On the second day, spread the matrix adhesive evenly on the bottom of the 24-well plate at a rate of 100 μL / well (operate on ice). Be careful to avoid air bubbles. Place it in a 4°C refrigerator for 15 minutes, and then transfer it to a 37°C constant temperature incubator for 30 minutes to allow it to solidify.

[0098] 3. Wash HUVEC cells with PBS (passage 2), digest the treated cells with trypsin (1.5 min), centrifuge at 1000 rpm for 5 min, discard the supernatant, resuspend in 2 mL of 5% ECM (Sciencell) medium, count the cells, and divide at 10 cells per well.4 Cells were evenly seeded onto the plate. A 5% FBS cell resuspension containing the drug was prepared: 900 μL of 250 mM 5% ECM, 100 μL of cell resuspension, and 1 μL of 250 mM 4-OI. The 24-well plate was removed from the substrate, and the cell suspension was slowly added to each well (100 μL per well). The plate was then incubated at 37°C.

[0099] 4. Photography: After incubation in an incubator for 4-6 hours, photographs were taken under an inverted microscope. Statistical analysis was performed using the tube formation analysis plugin of ImageJ.

[0100] The results are as follows Figure 11 As shown, intervention with 4-OI (250 μM) promotes the formation of luminal tubes in HUVECs.

[0101] In summary, the experimental results of Examples 1-7 confirm that octyl 4-itaconate can promote endothelial cell proliferation, migration, and scratch healing, which is beneficial to the recovery and reconstruction of damaged blood vessels; it can restore blood flow in the lower limb vessels after ischemia-reperfusion and significantly reduce the lesions in the lower limb muscle tissue.

[0102] The above detailed embodiments describe the implementation of the present invention; however, the present invention is not limited to the specific details described in the above embodiments. Within the scope of the claims and technical concept of the present invention, various simple modifications and changes can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

Claims

1. An application of octyl 4-itaconate, characterized in that, Used in the preparation of drugs that promote the proliferation, migration, or scratch healing of vascular endothelial cells; or used for purposes other than disease treatment and diagnosis that promote the proliferation, migration, or scratch healing of vascular endothelial cells.

2. The application according to claim 1, characterized in that, The octyl 4-itaconate is used to prepare drugs for relieving / treating peripheral vascular diseases.

3. The application according to claim 2, characterized in that, The dosage of the drug is 25-50 mg / kg based on the body weight of the organism.

4. The application according to claim 3, characterized in that, The concentration of octyl 4-itaconate in the drug is 5-8 mg / mL.

5. The application according to claim 2, characterized in that, The peripheral vascular disease includes peripheral vascular injury caused by lower limb ischemia-reperfusion.

6. The application according to claim 5, characterized in that, The peripheral vascular injury refers to poor blood circulation and tissue lesions.

7. The application according to any one of claims 1-6, characterized in that, The drug is available in oral or injectable form.

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