Application of kappa-opioid receptor stimulant U50488H in preparation of medicine for improving radioactive aortic vascular endothelial injury

Abstract

The invention discloses application of a kappa-opioid receptor stimulant U50488H in preparation of a medicine for improving radioactive aortic vascular endothelial injury, and belongs to the technical field of biological medicine. By selecting the kappa-opioid receptor stimulant with the (-)-U50488H isomer structure, the radioactive aortic vascular endothelial injury can be effectively improved. A plurality of experiments prove that the kappa-opioid receptor stimulant medicine containing the (-)-U50488H isomer structure is a medicine for slowing down aortic endothelial cell proliferation, inhibiting oxidative stress injury, improving protein level expression of Nrf2 / HO-1, improving nitric oxide level, improving endothelial nitric oxide synthase activity and reducing inflammatory response; when radioactive vascular endothelium is damaged, an Nrf2 / HO-1 path can be enhanced by activating a kappa-opioid receptor stimulant U50488H, so that the anti-oxidation and anti-inflammatory effects are achieved, and the vascular endothelium function of the kappa-opioid receptor stimulant U50488H is finally improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to the application of a κ-opioid receptor agonist U50488H in the preparation of a drug for improving radioactive aortic vascular endothelial injury. Background Art

[0002] Radiotherapy is a major method for cancer treatment. Epidemiological and clinical trial studies have shown that cardiovascular disease events in cancer patients receiving long-term radiotherapy are directly related to radiation exposure. For every 1 Gy increase in the average heart radiation dose, the risk of major coronary artery events increases by 7.4%.

[0003] Radiation-induced heart disease (RIHD) is one of the most common adverse consequences of radiotherapy and has become a major cause of death in long-term cancer survivors. RIHD is the result of the interaction of multiple complex pathways, including a series of heart diseases such as pericarditis, cardiomyopathy, coronary artery disease, vagal heart disease, and cardiac conduction abnormalities. Research suggests that the pathophysiological processes of most RIHD are related to endothelial cell injury and dysfunction. Currently, clinically, the occurrence of RIHD is mainly prevented by adjusting radiation dose and frequency, improving radiation targeting, reducing radiation area, etc. At the same time, drugs such as metformin and colchicine are considered to have a certain preventive effect on RIHD, but there is still a lack of large-scale and strong clinical evidence to support their benefits. Recent research has shown that modern technology cannot eliminate the risk of RIHD. Vascular endothelial injury and dysfunction, as a key link in the occurrence and development of RIHD, may become an important target for the prevention and treatment of RIHD.

[0004] Opioid Receptor (OR) belongs to the G protein-coupled receptor (GPCR) family and is mainly divided into three subtypes: μ (μ-Opioid Receptor, μ-OR), δ (δ-Opioid Receptor, δ-OR), and κ (κ-Opioid Receptor, κ-OR). The κ-opioid receptor is widely present in the cardiovascular system. The heart can produce and release endogenous κ-opioid peptides and act on κ-OR to regulate the activities of the cardiovascular system. Studies have found that κ-OR is involved in ischemic cardiac protection, activating κ-OR can prevent arrhythmia, reduce blood pressure, and reduce vascular remodeling. In short, activating κ-OR plays an obvious protective role in the cardiovascular system.

[0005] Nuclear factor-erythroid 2-related factor 2 (Nrf2) belongs to the Cap'n'Collar (CNC) family, and members of this family contain a conserved basic leucine zipper structure. The main function of Nrf2 is to activate the cellular antioxidant response by inducing the transcription of multiple genes, which can effectively counteract exogenous and endogenous harmful factors. Therefore, Nrf2 has traditionally been regarded as the main defense mechanism of cells and is considered an important regulator of cell survival. When cells are attacked by reactive oxygen species or electrophiles, Nrf2 rapidly translocates to the nucleus, forms a heterodimer with Maf proteins, and then binds to antioxidant response elements (AREs) to activate the expression of various antioxidant and anti-inflammatory factors. In addition, various protein kinases, such as mitogen-activated protein kinase (MAPK), protein kinase C (PKC), and phosphoinositide 3-kinase (PI3K), participate in the regulation of Nrf2 transcriptional activity by inducing Nrf2 phosphorylation. Hemeoxygenase (HO) is the key enzyme responsible for the degradation of endogenous iron protoporphyrin heme. There are three active subtypes of this enzyme: HO-1, HO-2, and HO-3. Among them, HO-1, also known as heat shock protein 32 (Hsp32), is an inducible subtype that can be activated by various stimuli such as ultraviolet light, infection, and heavy metals. As a key regulator of the expression of hemeoxygenase (HO-1), Nrf2 can regulate the expression of HO-1. Under the activation of Nrf2, HO-1 and its metabolites (including carbon monoxide, ferrous ions, and biliverdin) can effectively prevent the excessive oxidation of lipids and proteins by scavenging hydroxyl radicals, singlet oxygen, and superoxide anions, and play anti-inflammatory, antioxidant, and anti-apoptotic roles.

[0006] However, it is unclear whether activating κ-OR can effectively improve endothelial function during radioactive vascular endothelial injury and its effects on the expression and activity changes of eNOS and the content of NO; and whether, during radioactive vascular endothelial injury, activating κ-OR can enhance the Nrf2 / HO-1 pathway to exert antioxidant and anti-inflammatory effects and ultimately improve its vascular endothelial function is currently unclear. Summary of the Invention

[0007] The purpose of the present invention is to provide an application of the κ-opioid receptor agonist U50488H in the preparation of a drug for improving radioactive aortic vascular endothelial injury. The κ-opioid receptor agonist selected with the (-)-U50488H isomer structure can effectively improve radioactive aortic vascular endothelial injury.

[0008] The present invention is achieved through the following technical solutions:

[0009] Use of κ-opioid receptor agonist U50488H in the preparation of a drug for improving radioactive aortic vascular endothelial injury, wherein the κ-opioid receptor agonist U50488H is selected as the (-)-U50488H isomer structure.

[0010] The above (-)-U50488H has a specific chemical structure and can precisely bind specifically to the κ-opioid receptor. This binding has high affinity and selectivity, enabling (-)-U50488H to preferentially act on the κ-opioid receptor, while having weak or no obvious effect on other types of opioid receptors (such as μ-opioid receptor, δ-opioid receptor). This isomer can more effectively activate the κ-opioid receptor and trigger obvious pharmacological effects.

[0011] In activating the signaling pathway: when (-)-U50488H binds to the κ-opioid receptor, it will cause a conformational change in the receptor, thereby activating the G protein coupled to it. The activated G protein will further trigger a series of intracellular signal transduction events, mainly including inhibiting the activity of adenylate cyclase, reducing the production of the intracellular second messenger cyclic adenosine monophosphate (cAMP). At the same time, it can also regulate ion channels, such as promoting the outflow of potassium ions, hyperpolarizing the cell membrane, reducing the excitability of neurons; inhibiting the influx of calcium ions and reducing the release of neurotransmitters.

[0012] Preferably, the κ-opioid receptor agonist U50488H also includes two isomer structures of (+)-U50488H and (±)-U50488H.

[0013] Preferably, the drug is a drug for slowing down the proliferation of aortic endothelial cells, inhibiting oxidative stress injury, increasing the protein level expression of Nrf2 / HO-1, increasing the nitric oxide level, increasing the activity of endothelial nitric oxide synthase, and reducing the inflammatory response.

[0014] Preferably, the inhibition of oxidative stress injury is a decrease in the level of reactive oxygen species in the cells.

[0015] Preferably, the reduction of the inflammatory response is a decrease in the protein expression level of the inflammatory factor IL-6.

[0016] A drug for improving radioactive aortic vascular endothelial injury, wherein the drug is a drug composition with the (-)-U50488H isomer structure of the κ-opioid receptor agonist as the sole active ingredient or containing the (-)-U50488H isomer structure of the κ-opioid receptor agonist.

[0017] Preferably, the drug is composed of the (-)-U50488H isomer structure of the κ-opioid receptor agonist and pharmaceutically acceptable excipients.

[0018] Preferably, the drug is formulated into a clinically acceptable dosage form.

[0019] Preferably, the dosage form includes any one of decoction, powder, pill, medicinal liquor, lozenge, glue, and plaster.

[0020] Compared with the prior art, the present invention has at least the following technical effects:

[0021] The present invention provides an application of a κ-opioid receptor agonist U50488H in the preparation of a drug for improving radioactive aortic vascular endothelial injury. The κ-opioid receptor agonist selected with the (-)-U50488H isomer structure can effectively improve radioactive aortic vascular endothelial injury.

[0022] The κ-opioid receptor agonist U50488H has been verified through multiple experiments. The drug containing the κ-opioid receptor agonist with the (-)-U50488H isomer structure is a drug for slowing down aortic endothelial cell proliferation, inhibiting oxidative stress injury, increasing the protein level expression of Nrf2 / HO-1, increasing nitric oxide level, increasing endothelial nitric oxide synthase activity, and reducing inflammatory response. And when there is radioactive vascular endothelial injury, it can enhance the antioxidant and anti-inflammatory effects and ultimately improve its vascular endothelial function by activating the κ-opioid receptor agonist U50488H to enhance the Nrf2 / HO-1 pathway. Therefore, this technology provides new scientific basis and data support for the treatment and improvement of radioactive aortic vascular endothelial injury. Description of the Drawings

[0023] Figure 1 Schematic diagram of the effect of irradiation on the proliferation of mouse aortic endothelial cells;

[0024] Figure 2 Schematic diagram of the results of the cytotoxicity experiment of each drug;

[0025] Figure 3 Schematic diagram of the κ-OR agonist inhibiting oxidative stress injury of mouse aortic endothelial cells, where (A) is the ROS fluorescence intensity of each group under the fluorescence microscope; (B) is the analysis chart of the relative ROS fluorescence intensity;

[0026] Figure 4 Schematic diagram of the κ-OR agonist improving the inflammatory response of mouse aortic endothelial cells;

[0027] Figure 5 Schematic diagram of the change in the NO level and NOS activity of the κ-OR agonist improving mouse aortic endothelial cells;

[0028] Figure 6 Schematic diagram of the κ-OR agonist increasing the protein expression level of Nrf2 / HO-1 in irradiated cells. Detailed Embodiments

[0029] The following will describe the implementation scheme of the present invention in detail in combination with embodiments. However, those skilled in the art will understand that the following embodiments are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention. For the specific conditions not specified in the embodiments, they are carried out according to the conventional conditions or the conditions recommended by the manufacturer. For the reagents or instruments not specified for the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0030] The technical solution of a specific implementation manner of the present invention is as follows:

[0031] All U50488H described in the following experiments are in the (-)-U50488H structure.

[0032]

[0033] 1. Verify the effect of κ-opioid receptor agonists on improving radioactive vascular endothelial injury

[0034] 1.1 First, a radioactive arterial endothelial cell injury model was constructed to explore the cell proliferation under different irradiation doses.

[0035] 1.2 Construction of a radioactive vascular endothelial injury animal model:

[0036] The animal experiment was reviewed and approved by the Medical Ethics Committee of Chengdu Medical College (Cheng Yi Dong Lun

[2024] No. 067). All animal experiments were carried out in the SPF-class animal house of the Central Laboratory of Chengdu Medical College.

[0037] Based on the consideration of test power and the economy of experimental animals, 9 male C57BL / 6J mice at 7 weeks of age were randomly grouped in this project. During the experiment, all mice were fed solid feed. After 1 week of adaptive feeding, they were randomly divided into 3 groups, with 3 mice in each group.

[0038] The specific grouping is as follows: 1) Sham irradiation group: normal diet and water; 2) Irradiation group: a radioactive injury mouse model was established by X-ray irradiation; 3) Irradiation + U50488H (κ-OR agonist, intraperitoneal injection, 1.5 mg / kg) group.

[0039] The irradiation + U50488H group was intraperitoneally injected with the drug, and the other groups were intraperitoneally injected with an equal amount of normal saline. They were raised in a suitable environment for 1 week. After 1 week, the irradiation group and the irradiation + U50488H group received single-dose local heart and neck vessel irradiation, and the rest of the body was shielded with lead plates. The X-ray dose was 15 Gy (200 kV, 10 mA). On the 30th day after the radiation ended, the bilateral carotid arteries and aortas of the mice in each group were collected for the next experiment.

[0040] 1.3 The MAEC cells in good growth state were seeded at 1×10 per well4 Cells were re-seeded at a cell density into a 96-well cell culture plate, and the culture plate was pre-incubated in an incubator for 24 hours (37 °C, 5% CO2) to allow them to adhere. The cells were divided into: 0 Gy group, 5 Gy group, 10 Gy group, and 15 Gy group. After adhesion, they were respectively irradiated with a single dose of X-rays at doses of 0 Gy, 5 Gy, 10 Gy, and 15 Gy (200 kV, 10 mA). The proliferation of the cells was evaluated by the CCK-8 method at 1 day, 2 days, and 3 days after irradiation respectively.

[0041] After 1.4, the intervention experiments on endothelial cells were carried out using the κ-opioid receptor agonist U50488H, the Nrf2 specific inhibitor ML385, the HO-1 specific inhibitor ZnPP-IX, and the eNOS specific inhibitor L-NAME.

[0042] 1.5 As Figure 1 shown, it is a schematic diagram of the effect of irradiation on the proliferation of mouse aortic endothelial cells. Note: The data are expressed as ratios normalized to the results of cells from sham-irradiated control mice. *, **, ***, **** indicate statistically significant differences (***p < 0.001; ****p < 0.0001).

[0043] The results combined Figure 1 showed that with the increase of the irradiation dose, the proliferation of aortic endothelial cells slowed down significantly.

[0044] 2. Cytotoxicity experiment

[0045] To ensure the safety and effectiveness of the experiment, the CCK-8 method was used to conduct a drug toxicity experiment on the cells.

[0046] 2.1 Experimental method:

[0047] The CCK-8 method was used to detect the effect of different drug concentrations on cell viability. Before the experiment, relevant literature was consulted. For MACE, the optimal drug concentration of U50488H was 70 μmol / L, the optimal drug concentration of ML385 was 5 μmol / L, the optimal drug concentration of ZnPP-IX was 0.5 μmol / L, and the optimal drug concentration of L-NAME was 1×10 -4 mol / L. Based on the literature, the drug concentration of U50488H was set at 0, 30, 50, 70, 90, 110 μmol / L, the drug concentration of ML385 was set at 0, 4, 5, 10, 15, 20 μmol / L, the drug concentration of ZnPP-IX was set at 0, 0.2, 0.5, 1, 1.5 μmol / L, and the drug concentration of L-NAME was set at 0, 25, 50, 100, 150, 200 μmol / L. Each group had 3 replicate wells.

[0048] After digesting the well-growing MACE cells, inoculate them again in a 96-well cell culture plate at a cell density of 1×10 4 cells per well. Place the culture plate in an incubator and culture for 24 hours (37°C, 5% CO2) to allow them to adhere. Add media containing different concentrations of drugs and culture for 24h and 48h respectively. After sufficient culture time, add 10 μl of CCK-8 solution to each well and continue to culture for 1h in the dark. Measure the absorbance value (A) of each well at a wavelength of 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader. Calculate the cell viability (%) of each group according to the formula to screen the optimal drug concentration for subsequent experiments. Cell viability calculation formula:

[0049] Cell viability (%) = [A (experimental group) - A (blank group)] / [A (negative control group) - A (blank group)] × 100%

[0050] 2.2 Experimental results:

[0051] As Figure 2 shown, it is a schematic diagram of the cytotoxicity experimental results of each drug. Note: U: κ-opioid receptor agonist U50488H; M: Nrf2 specific inhibitor ML385; Z: HO-1 specific inhibitor ZnPP-IX; L: eNOS specific inhibitor L-NAME.

[0052] Combined Figure 2 results show that at the set drug concentrations, cells were treated for 24h or 72h respectively, and the cell activity was not significantly affected. The concentrations of the relevant drugs are as follows: U50488H: 70 μmol / L, ML385: 5 μmol / L, ZnPP-IX: 0.5 μmol / L, L-NAME: 1×10 -4 μmol / L. As Figure 2 shown, the selected drug concentrations have no significant toxicity to cells.

[0053] 3. Cellular ROS detection experiment

[0054] Subsequently, an assessment experiment was conducted on the oxidative stress of radioactive endothelial cell injury.

[0055] 3.1 Experimental method:

[0056] The DCFH-DA method was used to detect the ROS (reactive oxygen species) level in cells. It was divided into 0 Gy group, 10 Gy group, 10 Gy + U50488H group, 10 Gy + U50488H + ML385 group, 10 Gy + U50488H + ZnPP-IX group, and 10 Gy + U50488H + L-NAME group.

[0057] After digesting the well-growing MAEC cells, inoculate them again in a 96-well cell culture plate at a cell density of 1×104 The cells were re-seeded at a cell density into a 96-well cell culture plate, and the culture plate was placed in an incubator for 24 hours (37 °C, 5% CO2) to allow them to adhere.

[0058] Then, high-glucose media containing drugs were prepared separately. The specific drug concentrations were as follows: κ-OR agonist U50488H at 70 μmol / L, Nrf2-specific inhibitor ML385 at 5 μmmol / L, HO-1-specific inhibitor ZnPP-IX at 0.5 μmmol / L, and eNOS-specific inhibitor L-NAME at 1×10 -4 mmol / L. The cells were cultured with the corresponding media for 48 h.

[0059] Groups of 10 Gy, 10 Gy + U50488H, 10 Gy + U50488H + ML385, 10 Gy + U50488H + ZnPP-IX; and 10 Gy + U50488H + L-NAME received single X-ray irradiation at a dose of 10 Gy (200 kV, 10 mA). Cells were collected 4 h after irradiation for ROS detection.

[0060] The detection steps were as follows: The DCFH-DA probe was diluted 1:1000 with serum-free culture medium to prepare a working solution with a final concentration of 10 μmol / L. After aspirating the old culture medium in the cell plate with a pipette, 100 μl of the diluted DCFH-DA working solution was added to each well, and it was incubated in the 37 °C cell culture incubator in the dark for 20 min. The cells were washed 3 times with serum-free cell culture medium to fully remove the DCFHDA that had not entered the cells.

[0061] Then, Hoechst33342 staining solution was used for nuclear staining: The culture medium was removed, 100 μl of the staining solution was added to each well, and it was placed in the dark at room temperature for 10 min. The staining solution was aspirated, and then washed 3 times with PBS, 3 min each time, to remove the excess dye.

[0062] Finally, a fluorescence microscope and the analysis software Image J were used to quantitatively analyze the fluorescence in the cells to evaluate the ROS content.

[0063] 3.2 Experimental results:

[0064] As Figure 3As shown, it is a schematic diagram of κ-OR agonist inhibiting oxidative stress injury in mouse aortic endothelial cells. Among them, (A) is the ROS fluorescence intensity of each group under a fluorescence microscope. The scale bar is 20 μm; (B) is the analysis chart of relative ROS fluorescence intensity. Note: U: κ-opioid receptor agonist U50488H; M: ML385; Z: ZnPP-IX; L: L-NAME. Mouse aortic endothelial cells were irradiated with X-rays (10 Gy) or sham-irradiated (0 Gy), and their induction effect on ROS was observed. The data were expressed as the ratio normalized to the ROS results of the sham-irradiated control mouse cells. *, **, ***, **** indicate statistically significant differences (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001).

[0065] Combined Figure 3 It can be seen from the results that 70 μmol / L of U50488H was selected in the experiment. Compared with the 0 Gy group, the ROS level in the 10 Gy group increased significantly (P < 0.001); when the κ-OR agonist was introduced, the ROS level in the cells decreased (P < 0.01). Compared with the 10 Gy + U group, after introducing the Nrf2 specific inhibitor ML385, the HO-1 specific inhibitor ZnPP-IX, and the eNOS specific inhibitor L-NAME, namely the 10 Gy + U + M group, the 10 Gy + U + Z group, and the 10 Gy + U + L group, the ROS levels all increased (P < 0.01, P < 0.01, P < 0.001). There was no statistical significance between the 10 Gy + U + M group and the 10 Gy + U + Z group.

[0066] Therefore, the κ-OR agonist exerts an antioxidant stress effect through the Nrf2 / HO-1 pathway.

[0067] 4. Detection of IL-6 expression by Western-Blot

[0068] 4.1 Experimental method:

[0069] (1) Cell culture: Seed well-growing MAEC cells in a 6-well plate; (2) Total protein extraction: ① Prepare cell lysate; ② Lyse the cells; ③ Extract: Pre-cool the centrifuge in advance. After the cells are fully lysed, centrifuge at 12,000 rpm and 4 °C for 10 min. Immediately transfer the supernatant to a new pre-cooled 1.5 ml EP tube, which is the cytoplasmic protein. (3) Determine the total protein concentration of cells by BCA method; (4) Polyacrylamide gel electrophoresis: ① Prepare 10% separating gel; ② Prepare 5% stacking gel; ③ Load the sample; ④ Electrophoresis. (5) Membrane transfer: ① Prepare the membrane transfer solution; ② Treat the gel: Cut the gel to an appropriate size according to the marker, remove the stacking gel and unnecessary regions, and place the gel in the membrane transfer solution; ③ Prepare the "sandwich"; ④ Membrane transfer. (6) Blocking; (7) Immunoblotting reaction: Dilute the primary antibody in a certain proportion. Take out the blocked PVDF membrane strip and immediately add the corresponding diluted primary antibody. Incubate overnight on a shaker at 4 °C. Discard the primary antibody and wash the membrane strip 3 times with 1×TBST buffer on a shaker at room temperature for 5 min each time. Add the secondary antibody diluted at a ratio of 1:5000 and incubate at room temperature for 2 h. Discard the secondary antibody and wash the membrane 3 times with 1×TBST buffer for 10 min each time. (8) Color development and photography; (9) Quantify protein expression using ImageJ software.

[0070] 4.2 Experimental results:

[0071] Figure 4 As shown, the κ-OR agonist improves the inflammatory response of mouse aortic endothelial cells. Note: (A) Immunoblot of IL-6 protein expression changes; (B) Statistical analysis of relative protein expression levels. U: κ-opioid receptor agonist U50488H; M: ML385; Z: ZnPP-IX; L: L-NAME. *, ** indicate statistically significant differences (*P < 0.05; **P < 0.01; ns: no difference between the two groups).

[0072] Compared with the 0 Gy group, the IL-6 protein level in the 10 Gy group increased significantly after irradiation (P < 0.01), while the IL-6 protein expression level in the group treated with the κ-OR agonist decreased (P < 0.01). Compared with the 10 Gy + U group, the IL-6 expression levels in the 10 Gy + U + M group, 10 Gy + U + Z group, and 10 Gy + U + L group increased (P < 0.01, P < 0.01, P < 0.01). There was no statistically significant difference between the 10 Gy + U + M group and the 10 Gy + U + Z group.

[0073] The results showed that the κ-OR agonist inhibited the expression of inflammatory factors in irradiated cells by activating the Nrf2 / HO-1 pathway.

[0074] 5. NO and NOS activity detection experiment

[0075] κ-OR Agonist Improves NO Level and NOS Activity in Mouse Aortic Endothelial Cells

[0076] Experimental Methods:

[0077] 5.1 Detection of NO Content: Cells were treated according to the cell treatment method in the ROS detection experiment. The experiment was carried out 4 h after irradiation. Cells were lysed using cell and tissue lysis buffer. The standards in the kit were diluted with cell culture medium to 0, 1, 2, 5, 10, 20, 40, 60, 100 μmol / L. 50 μL of Griess Reagent I and Griess Reagent II were added to each well respectively. The absorbance was measured at 540 nm using an enzyme-linked immunosorbent assay (ELISA) reader. The standard curve was plotted based on the absorbance of the standards, and then the concentration of nitric oxide in the samples was calculated according to the standard curve of the standards.

[0078] 5.2 Detection of NOS Activity: Visible light spectrophotometry was used for detection. The cells in each group were added with extraction solution according to the requirements of the kit, and were sonicated in an ice bath (power: 200 W, sonication for 3 s, interval for 7 s, total time: 5 min), and then centrifuged at 4 °C, 12,000 g for 15 min. The precipitate was discarded, and the supernatant was placed on ice. The corresponding extraction solution was added, mixed well, and left standing at room temperature for 10 min. 1 mL of the reaction solution was taken and placed in a 1 mL glass cuvette to measure the absorbance at 550 nm for each tube. Finally, the activity of NOS was calculated according to the formula.

[0079] 5.3 Experimental Results:

[0080] As Figure 5 shown, the changes in NO levels and NOS activities in each group after irradiation and κ-OR agonist treatment are presented. Note: (A) Detection of NO level in cells 4 h after irradiation by enzymatic method; (B) Analysis of TNOS activity; (C) Analysis of iNOS activity; (D) Analysis of eNOS activity. U: κ-opioid receptor agonist U50488H; NO: nitric oxide; TNOS: total nitric oxide synthase; iNOS: inducible nitric oxide synthase; eNOS: endothelial nitric oxide synthase; M: ML385; Z: ZnPP-IX; L: L-NAME. The data are expressed as the ratio normalized to the results of the sham-irradiated control mouse cells. *, **, ***, **** indicate statistically significant differences (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001).

[0081] Combined with Figure 5As can be seen from the results in A, compared with the 0 Gy group, the NO level in the 10 Gy group of cells decreased significantly (14.22 ± 0.72 μmol / gprot VS. 3.56 ± 0.81 μmol / gprot, P < 0.0001). After using the κ-OR agonist, the NO level in the cells increased somewhat (3.56 ± 0.81 μmol / gprot VS. 11.75 ± 0.45 μmol / gprot, P < 0.0001).

[0082] Combined with Figure 5 As can be seen from the results in B, compared with the 10 Gy + U group, the NO levels in the 10 Gy + U + M group, 10 Gy + U + Z group, and 10 Gy + U + L group all decreased significantly (P < 0.0001 for all).

[0083] Combined with Figure 5 As can be seen from the results in C, there was no statistical significance when comparing the 10 Gy + U + M group with the 10 Gy + U + Z group.

[0084] Combined with Figure 5 As can be seen from the results in D, the activity of eNOS also showed the same trend: compared with the 0 Gy group, the eNOS activity in the 10 Gy group of cells decreased significantly (P < 0.01), and after introducing the κ-OR agonist, the eNOS activity increased somewhat (P < 0.01).

[0085] Compared with the 10 Gy + U group, the eNOS activities in the 10 Gy + U + M group, 10 Gy + U + Z group, and 10 Gy + U + L group all decreased (P < 0.001, P < 0.001, P < 0.05).

[0086] Therefore, the effect of the κ-OR agonist in improving the NO level and NOS activity in irradiated cells is exerted through the Nrf2 / HO-1 pathway.

[0087] 6. Detection of the protein expression levels of Nrf2 and HO-1 by Western-Blot

[0088] The κ-OR agonist can increase the protein expression levels of Nrf2 and HO-1 in irradiated cells.

[0089] 6.1 Experimental method:

[0090] (1) Cell culture: Seed well-growing MAEC cells in a 6-well plate; (2) Total protein extraction: ① Prepare cell lysate; ② Lyse the cells; ③ Extract: Pre-cool the centrifuge in advance. After the cells are fully lysed, centrifuge at 12,000 rpm and 4 °C for 10 min, and immediately transfer the supernatant to a new pre-cooled 1.5 ml EP tube, which is the cytoplasmic protein. (3) Determine the total protein concentration of the cells by the BCA method; (4) Polyacrylamide gel electrophoresis: ① Prepare 10% separating gel; ② Prepare 5% stacking gel; ③ Load the sample; ④ Electrophoresis. (5) Transfer membrane: ① Prepare transfer buffer; ② Treat the gel: Cut the gel to an appropriate size according to the marker, remove the stacking gel and unnecessary regions, and put the gel into the transfer buffer; ③ Prepare the "sandwich"; ④ Transfer the membrane. (6) Blocking; (7) Immunoblotting reaction: Dilute the primary antibody in a certain proportion. Take out the blocked PVDF membrane strip, immediately add the corresponding diluted primary antibody, and place it on a shaker at 4 °C and incubate overnight. Discard the primary antibody, wash the membrane strip 3 times with 1×TBST buffer on a shaker at room temperature for 5 min each time. Add the secondary antibody diluted at a ratio of 1:5000, incubate at room temperature for 2 h, discard the secondary antibody, and wash the membrane 3 times with 1×TBST buffer for 10 min each time. (8) Develop color and take pictures; (9) Quantify protein expression with ImageJ software.

[0091] 6.2 Experimental results:

[0092] Using β-actin as an internal reference, combined with Figure 6 Figure B in

[0093] Combined with Figure 6Among them, C represents the change in HO-1 protein level. Compared with the non-irradiated group, the expression level of HO-1 increased after receiving 10 Gy irradiation (P < 0.05). Compared with the 10 Gy group, in the 10 Gy + U group, the HO-1 expression level increased (P < 0.0001). Compared with the 10 Gy + U group, the protein expression levels in the 10 Gy + U + M group and the 10 Gy + U + Z group were significantly decreased (P < 0.001, P < 0.0001). There was no significant difference in the HO-1 protein expression level between the 10 Gy + U + M group and the 10 Gy + U + Z group.

[0094] In summary, activating κ-OR can increase the expression levels of Nrf2 and HO-1 proteins in cells after irradiation.

[0095] Finally, it should be noted that the above are only the preferred embodiments of the present invention and are not intended to limit the protection scope of the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. Use of κ-opioid receptor agonist U50488H in the preparation of a drug for improving radioactive aortic vascular endothelial injury, characterized in that, The κ-opioid receptor agonist U50488H uses the (-)-U50488H isomer structure.

2. Use of a kappa-opioid receptor agonist U50488H according to claim 1 in the preparation of a drug for improving radioactive aortic vascular endothelial injury, characterized in that, The κ-opioid receptor agonist U50488H also includes two isomer structures of (+)-U50488H and (±)-U50488H.

3. Use of a kappa-opioid receptor agonist U50488H according to claim 1 in the preparation of a drug for improving radioactive aortic vascular endothelial injury, characterized in that, The drug is a drug that slows down the proliferation of aortic endothelial cells, inhibits oxidative stress damage, increases the protein level expression of Nrf2 / HO-1, increases the nitric oxide level, increases the activity of endothelial nitric oxide synthase, and reduces the inflammatory response.

4. Use of a kappa-opioid receptor agonist U50488H according to claim 3 in the preparation of a drug for improving radioactive aortic vascular endothelial injury, characterized in that, The inhibition of oxidative stress damage is a decrease in the level of reactive oxygen species in cells.

5. Use of a kappa-opioid receptor agonist U50488H according to claim 3 in the preparation of a drug for improving radioactive aortic vascular endothelial injury, characterized in that, The reduction of the inflammatory response is a reduction in the protein expression level of the inflammatory factor IL-6.

6. A drug for improving endothelial injury of the radioactive aorta, characterized in that, The drug uses the (-)-U50488H isomer structure of the κ-opioid receptor agonist described in claim 1 as the sole active ingredient or is a pharmaceutical composition containing the (-)-U50488H isomer structure of the κ-opioid receptor agonist.

7. The drug for improving radioactive aortic vascular endothelial injury according to claim 6, characterized in that, The drug is composed of the (-)-U50488H isomer structure of the κ-opioid receptor agonist and pharmaceutically acceptable excipients.

8. The drug for improving radioactive aortic vascular endothelial injury according to claim 6, characterized in that, The drug is prepared into a clinically acceptable dosage form.

9. The drug for improving radioactive aortic vascular endothelial injury according to claim 8, characterized in that, The dosage form includes any one of decoctions, powders, pills, medicinal wines, lozenges, gelatin, and ointments.

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