Application of 12-HEPE in preparation of medicine for treating hypertension

Abstract

The invention discloses application of 12-HEPE in preparation of a medicine for treating hypertension, and belongs to the field of biological medicine. The invention provides application of 12-hydroxyeicosapentaenoic acid (12-HEPE) in the aspect of blood pressure regulation and control for the first time. Experimental results show that the 12-HEPE can significantly reduce systolic pressure and diastolic pressure of hypertension modeling mice, significantly improve kidney and blood vessel lesions and dysfunction caused by hypertension, and relieve oxidative stress injury of lesion tissues. The invention provides a new optional medicine for preventing and treating hypertension and maintaining the healthy level of blood pressure, and 12-HEPE has a comprehensive and remarkable curative effect on hypertension and complications thereof. The invention has prominent practical application value and wide application prospect in the medical industry.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to the use of 12-HEPE in the preparation of drugs for treating hypertension. Background Technology

[0002] With the development of the times, the number of patients with hypertension continues to increase. According to survey data, among patients who have received antihypertensive drug treatment, the blood pressure control rate is only 37.6%. Existing drugs (such as ACEIs, ARBs, and calcium channel blockers) have side effects such as dry cough, edema, and electrolyte imbalance, and there is an urgent need to develop new and safe drugs.

[0003] Clinical data have revealed a positive correlation between free fatty acids (FFA) and blood pressure. Furthermore, FFA metabolism influences the development and progression of hypertension by inducing vascular dysfunction and activating the RAAS system. 12-Hydroxyeic acid (12-HEPE) is an omega-3 fatty acid metabolite produced from EPA (eicosapentaenoic acid) through the metabolism of 12-lipoxygenase (12-LOX). 12-HEPE plays an important role in various physiological and pathological processes, including regulating inflammatory responses and metabolic processes. Studies have also found that 12-HEPE effectively protects spermatogonia by targeting GPR120 and activating downstream signaling pathways. Whether 12-HEPE can be a potential beneficial drug component in hypertension and its regulatory mechanism have not yet been reported. Summary of the Invention

[0004] The purpose of this invention is to provide the application of 12-HEPE in the preparation of drugs for treating hypertension, in order to solve the problems existing in the prior art. This invention provides a new drug option for the prevention, treatment and maintenance of healthy blood pressure levels for hypertension. 12-HEPE has comprehensive and significant therapeutic effects on hypertension and its complications. This invention has outstanding practical application value and broad application prospects in the medical industry.

[0005] To achieve the above objectives, the present invention provides the following solution:

[0006] This invention provides the application of 12-hydroxyeicosapate in the preparation of health foods that help maintain healthy blood pressure levels.

[0007] This invention also provides a health food that helps maintain healthy blood pressure levels, with 12-hydroxyeicosapentaenoic acid as the main active ingredient.

[0008] Furthermore, this also includes food science-acceptable additives.

[0009] The present invention also provides the use of 12-hydroxyeicosapride in the preparation of medicaments for the prevention and / or treatment of hypertension.

[0010] The present invention also provides a drug for the prevention and / or treatment of hypertension, with 12-hydroxyeicosapriaenoic acid as the main active ingredient.

[0011] Furthermore, it also includes pharmaceutically acceptable excipients.

[0012] The present invention also provides the application of 12-hydroxyeicosapate in the preparation of a drug for treating complications of hypertension.

[0013] Furthermore, the hypertension complications include hypertensive nephropathy and hypertensive vascular disease.

[0014] The present invention also provides a drug for treating complications of hypertension, with 12-hydroxyeicosalicylic acid as the main active ingredient.

[0015] Furthermore, it also includes pharmaceutically acceptable excipients.

[0016] The present invention discloses the following technical effects:

[0017] This invention provides the first application of 12-hydroxyeicosapentaenoic acid (12-HEPE) in blood pressure regulation. Experimental results show that 12-HEPE can significantly reduce systolic and diastolic blood pressure in hypertensive model mice, significantly improve kidney and vascular lesions and dysfunction caused by hypertension, and alleviate oxidative stress damage in diseased tissues. This invention provides a new drug option for the prevention, treatment, and maintenance of healthy blood pressure levels in hypertension. 12-HEPE has comprehensive and significant therapeutic effects on hypertension and its complications. This invention has outstanding practical application value and broad application prospects in the medical industry. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 Statistical graph showing the effect of hypertension on FFA metabolic disorder and decreased 12-HEPE levels; where A represents human serum FFA level; B represents mouse serum FFA level; C represents human serum 12-HEPE level; D represents mouse serum 12-HEPE level; *P<0.05, **P<0.01, ***P<0.001; n≥5;

[0020] Figure 2Statistical graph showing the effect of 12-HEPE on blood pressure levels in hypertensive mice; where A represents systolic blood pressure and B represents diastolic blood pressure; *P<0.05, ***P<0.001, ****P<0.0001; n≥5;

[0021] Figure 3 Figure 1 shows the experimental results of 12-HEPE alleviating kidney lesions in hypertensive mice; A is H&E staining to observe glomerular morphology; B is Sirius red staining of the kidney to reflect collagen deposition; C is Masson staining of the kidney to reflect the degree of fibrosis; D is DHE immunofluorescence staining to reflect the renal ROS production; Scale bar: 25 μm; ***P<0.001, ****P<0.0001; n≥6;

[0022] Figure 4 Figure 1 shows the results of an experiment on 12-HEPE-induced vascular lesions in hypertensive mice. A shows H&E staining to observe aortic morphology; B shows Sirius red staining of the aorta to reflect collagen deposition; C shows DHE immunofluorescence staining to reflect ROS generation in the aorta; D shows α-SMA immunofluorescence staining to reflect aortic fibrosis. Scale bar: 25 μm; *P<0.05, **P<0.01, ****P<0.0001; n≥6.

[0023] Figure 5 Figure 1 shows the results of 12-HEPE improving vascular dysfunction; where A is the endothelium-dependent (acetylcholine, Ach) concentration-relaxation curve of the abdominal aorta; B is the non-endothelial cell-dependent (sodium nitroprusside, SNP) concentration-relaxation curve of the abdominal aorta; *P<0.05; n≥6;

[0024] Figure 6 Figure 1 shows the results of 12-HEPE upregulating FFAR4 expression in the kidneys of hypertensive mice; where A is the Western blot (WB) results of FFAR4 and GSTA3 protein in mouse kidneys; B is the protein level statistical graph, **P<0.01, ***P<0.001; n≥3. Detailed Implementation

[0025] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0026] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0027] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0028] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0029] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0030] The 12-HEPE of this invention is 12-hydroxyeicosapride, with the molecular formula C2. 20 H 30 O3, CAS number: 116180-17-7.

[0031] Example 1

[0032] 1. Experimental Methods

[0033] 1.1 Establishment of an Ang II (angiotensin II)-induced mouse model of hypertension

[0034] (1) Animal experimental grouping

[0035] Eight-week-old control groups with the same genetic background (WT), hypertension control group (WT-Ang II), and experimental group (WT-Ang II+12-HEPE) were selected.

[0036] (2) Drug preparation

[0037] Ang II (dose 490 ng / kg / min): In a sterile laminar flow hood, dissolve 10 mg of Ang II in 1 mL of PBS, mix thoroughly, dispense into 500 μL tubes, and store the stock solution at -20°C.

[0038] 12-HEPE (200 μg / kg / 24h intraperitoneal injection): diluted with PBS.

[0039] (3) Handling of laboratory animals

[0040] Five days of acclimatization training were conducted in mice using the tail-crotch method to measure blood pressure. All animals were fasted for 12 hours prior to modeling but allowed free access to water. On the day of modeling, the experimental group mice were weighed, and the required dose of Ang II was calculated based on their weight. The required dose of Ang II was mixed evenly with sterile PBS and injected into the osmotic pump (the osmotic pump was incubated overnight in sterile PBS at 37°C). After anesthetizing the mice with isoflurane, the skin in the scapular region was shaved and disinfected. A transverse incision of appropriate width was made in the scapular region using aseptic surgical techniques. The skin and tissue were separated using hemostatic forceps, and the osmotic pump corresponding to the mouse's weight was implanted subcutaneously (pump head facing the mouse's head). The wound was sutured and disinfected to prevent infection. The control group mice underwent a sham surgery experiment with an osmotic pump containing an equal amount of PBS.

[0041] (4) Animal sampling

[0042] Blood pressure was monitored every other day. Mice were harvested 14 days later. Mice were starved for 12 hours beforehand. Serum was collected by cardiac blood sampling. The serum was allowed to stand at room temperature for 30 minutes and then centrifuged at 3000 rpm for 10 minutes at 4°C using a low-temperature ultracentrifuge. The supernatant was collected and stored at -80°C. Other organs were removed, weighed, and a portion of the tissue was fixed in tissue fixative for subsequent paraffin embedding. The remaining portion was stored in cryovials at -80°C for long-term preservation. Blood vessels and perivascular fat were dissected under a stereomicroscope. The thoracic aorta was taken, embedded in an OCT scanner, and fixed at -20°C. The remaining blood vessel portion was stored in cryovials at -80°C for long-term preservation.

[0043] 1.2 Monitoring of blood pressure in the tail artery of mice

[0044] Mice were fed a normal diet and underwent blood pressure adaptation training using the tail-crotch method for 5 consecutive days. Systolic blood pressure (SBP), diastolic blood pressure (DBP), and heart rate (HR) were measured at the same time each day until the measurements were reproducible, excrement decreased, and the blood pressure waveform stabilized. Blood pressure was measured and recorded on days 2, 4, 6, 8, 10, 12, and 14 after administration of the Ang II sustained-release pump.

[0045] 1.3WB Experiment

[0046] 1.3.1 Extraction of proteins from tissues and cells

[0047] (1) Preparation of lysis buffer: Mix the tissue or cell protein extraction lysis buffer with PMSF at a volume ratio of 100:1 on ice.

[0048] (2) Preparation of tissue fluid or cell fluid: After sterilizing the blade and forceps, take an appropriate amount of kidney and adipose tissue, add lysis buffer and steel ball, and homogenize in a high-throughput homogenizer until there is no solid residue; discard the old culture medium in the cells, wash with PBS, add an appropriate amount of cell lysis buffer, and let stand on ice for 30 min. Use a cell scraper to scrape the cells into centrifuge tubes.

[0049] (3) Centrifugation: Centrifuge 3 times at 12000 rpm in a 4℃ ultracentrifuge, each time for 15 min, and aspirate the supernatant until it is clear and free of flocculent matter.

[0050] 1.3.2 Protein concentration determination (BCA method)

[0051] (1) Plotting the BCA standard curve

[0052] Add 20, 19, 18, 16, 12, 8, 4, and 0 μL of pure water to each well of a 96-well plate, followed by 0, 1, 2, 4, 8, 12, 16, and 20 μL of 0.5 mg / mL standard solution, for a total of 20 μL per well. Add 2 μL of homogenate from each sample tissue, followed by 18 μL of pure water, for a total of 20 μL per well, effectively diluting the sample 10-fold.

[0053] (2) Prepare BCA working solution at a volume ratio of 50:1, add 200 μL of working solution to each well, and incubate in a constant temperature oven at 37℃ for 30 min.

[0054] (3) Measure the OD value of each well at a wavelength of 570 nm using an enzyme-linked immunosorbent assay (ELISA) reader, and calculate the concentration of the tissue homogenate for each sample based on the OD value results.

[0055] (4) Prepare samples as needed. After the samples are prepared, mix them well, heat them in a constant temperature metal bath at 100℃ for 10 min, cool them on ice, centrifuge them in a 4℃ ultracentrifuge at 2000 rpm for 5 min, and store them in a -20℃ refrigerator for subsequent experiments.

[0056] 1.3.3 Electrophoresis and Electrotransfer

[0057] Table 1. Preparation of 10% Stacking Gel and Separating Gel

[0058]

[0059] (1) First install the glass plate, prepare the separating gel according to the formula, press it onto the top of the glass plate with isopropanol, and after the separating gel solidifies, pour off the upper layer of isopropanol, add the prepared 10% concentrated gel, and quickly insert the comb to wait for solidification.

[0060] (2) Electrophoresis: The protein sample was subjected to SDS-PAGE electrophoresis at 70V. After the protein markers on both sides were separated for about 30 minutes, the voltage was changed to 120V until the electrophoresis was completed.

[0061] (3) Transfer: Prepare the transfer buffer in advance and pre-cool it in a refrigerator at 4°C. Prepare a PVDF membrane of the same size as the gel and soak it in methanol for 5 minutes beforehand. After electrophoresis, remove the gel and transfer it to filter paper. Place the gel in the black and white transfer plates in the order of sponge pad, filter paper, and gel. Place the plate in the transfer tank, pour in the pre-cooled transfer buffer, set up the instrument, place it on ice, and perform electrophoresis at 270mA for 90 minutes.

[0062] (4) Blocking: Place the electroporated PVDF membrane with the protein side up and block it in 5% skim milk at room temperature for 90 min. Wash the milk three times with TBST buffer, 10 min each time.

[0063] (5) Antibody incubation: Cut the required molecular weight band, add the target antibody dilution solution, and incubate overnight on a shaker at 4°C. Recover the primary antibody and wash three times with TBST buffer for 10 min each time. Then add the secondary antibody with the corresponding properties, incubate at room temperature with shaking for 1 h, and wash three times with TBST buffer for 10 min each time.

[0064] (6) Exposure: Incubate with exposure solution for 1 minute and then expose.

[0065] (7) Quantitative statistics: Use software to perform grayscale statistical analysis on indicators and internal parameter bands.

[0066] 1.4 Paraffin sections and frozen sections

[0067] 1.4.1 Paraffin embedding and sectioning of kidney

[0068] (1) The tissue was soaked in 4% paraformaldehyde for 24 hours and then transferred to 75% ethanol for overnight soaking.

[0069] (2) Alcohol gradient dehydration: soak in 80% ethanol for 30 min, soak in 95% ethanol for 30 min, soak in 95% ethanol for 30 min, soak in 100% ethanol for 30 min, soak in 100% ethanol for 30 min.

[0070] (3) Transparent: Soak in xylene I for 1 hour, then soak in xylene II for 1 hour.

[0071] (4) Wax impregnation: Soak in soft wax for 1.5 hours, then transfer to hard wax and soak for 1.5 hours.

[0072] (5) Embedding: Embed the tissue block with the cut surface facing down.

[0073] (6) Tissue sections: Cut 5μm tissue sections and dry them in an oven at 65℃ for 4 hours for later use.

[0074] 1.4.2 Frozen embedding and sectioning of the thoracic aorta

[0075] (1) The thoracic aorta was separated from the perivascular fat, and a thoracic aorta of about 2 mm was placed vertically in an OCT and frozen at -20℃.

[0076] (2) Set the temperature of the cryostat to -25°C and place the slice box on ice.

[0077] (3) Use OCT to fix the tissue block on the clamping block, let it stand for about 5 minutes, adjust the angle, and use a thickness of 20μm to trim the slide until the tissue is intact.

[0078] (4) For the thoracic aorta, after the complete thoracic aorta section is cut out, adjust the slice thickness to 5 μm.

[0079] (5) Place the marked glass slides tightly onto the OCT slides and store them in a slide box at -20℃.

[0080] 1.5 Pathological staining

[0081] 1.5.1 Pretreatment of Paraffin Sections

[0082] Selected paraffin sections of mouse kidneys were placed on a baking machine to melt the wax. The sections were then placed in xylene solution I and allowed to stand for 15 minutes, followed by xylene solution II and allowed to stand for 15 minutes. Next, the sections were sequentially transferred to 100% anhydrous ethanol I for 5 minutes, 100% anhydrous ethanol II for 5 minutes, 95% ethanol solution for 5 minutes, and 80% ethanol solution for 5 minutes to wash away the xylene on the sections. The slides were then removed from the slide holder and placed in a pure water box to complete the dewaxing and rehydration process (during which the slides were prevented from drying out and affecting the morphology of the tissue staining).

[0083] 1.5.2 Pretreatment of frozen sections

[0084] Pre-cool the frozen sections in acetone at -20°C for 20 minutes; place the frozen sections on a slide rack and let them sit at room temperature for 5 minutes; then fix the sections in acetone for 15 minutes and let them evaporate at room temperature for 10 minutes until the acetone has completely evaporated; finally, place them in pure water for use.

[0085] 1.5.3 Masson staining

[0086] (1) Select pretreated paraffin sections of the kidney to be stained;

[0087] (2) Dye with Ponceau red dye for 2 minutes.

[0088] (3) Rinse with pure water.

[0089] (4) Weak acid differentiation for 1 min.

[0090] (5) Dye with phosphomolybdic acid dye for 1 minute.

[0091] (6) Dye with aniline blue dye for 2 minutes.

[0092] (7) Rinse with pure water.

[0093] (8) Weak acid differentiation for 1 min.

[0094] (9) Alcohol gradient dehydration: 95% alcohol for 5s, 100% ethanol I for 5s, 100% ethanol II for 1min.

[0095] (10) Transparent: Soak twice in xylene for 5 minutes each time.

[0096] (11) Seal the film with neutral resin liquid and take a picture.

[0097] 1.5.4 Sirius Red Staining

[0098] (1) Select pre-treated frozen sections of the thoracic aorta or paraffin sections of the kidney to be stained.

[0099] (2) Stain with Sirius red dye for 30 min and 45 min respectively.

[0100] (3) Alcohol dehydration: 100% ethanol for 3 seconds, 100% ethanol for 1 minute.

[0101] (4) Transparent: Xylene I 5 min; Xylene II 15 min.

[0102] (5) Mount the slide with neutral resin and observe it under a microscope.

[0103] 1.5.5 H&E staining

[0104] (1) Select pre-treated frozen sections of the thoracic aorta or paraffin sections of the kidney to be stained.

[0105] (2) Place the tissue in 100% ethanol for 5 min, 95% ethanol for 5 min, 85% ethanol for 5 min, 75% ethanol for 5 min, and tap water for 5 min.

[0106] (3) Soak the slices in hematoxylin for 5 minutes and rinse them with distilled water.

[0107] (4) Add differentiation solution to the tissue for 1 minute, rinse briefly with distilled water, and then rinse with running tap water for 10 minutes.

[0108] (5) Soak the slices in eosin staining for 3 minutes, then rinse briefly with tap water.

[0109] (6) 95% alcohol for 5s, 100% ethanol I for 10s, 100% ethanol II for 1min.

[0110] (7) Transfer the slice to xylene I for 30 min; xylene II for 30 min.

[0111] (8) Seal the film with neutral resin, let it air dry in a fume hood, and then take a film for observation.

[0112] 1.5.6 Immunohistochemical Experiment

[0113] Immunohistochemical staining (F4 / 80 index) was performed on paraffin sections of mouse kidneys.

[0114] (1) Antigen retrieval: The slides were subjected to antigen retrieval using 1×0.01mol / L sodium citrate buffer. First, the solution was brought to a boil in a microwave oven on high for 8 minutes, then heated on medium heat for 15 minutes on low heat. The sodium citrate solution was then allowed to cool naturally to room temperature. The slides were washed three times with PBST buffer for 5 minutes each time.

[0115] (2) Blocking endogenous peroxidase: Add 30 μL of endogenous peroxidase blocking agent solution to each tissue, incubate at room temperature for 30 min, and then wash 3 times with PBST buffer for 5 min each time.

[0116] (3) Add 30 μL of normal non-immune animal serum, incubate at room temperature for 30 min, and then wash 3 times with PBST buffer for 5 min each time.

[0117] (4) Add primary antibody solution: Add 50 μL of primary antibody solution to each tissue and incubate overnight at 4°C. After incubation, place at room temperature for 1 h and wash three times with PBST buffer for 5 min each time.

[0118] (5) Add secondary antibody detection reagent: Add 50 μL of the corresponding secondary antibody to each tissue, incubate at room temperature for 1 h, wash 3 times with PBST buffer, 5 min each time.

[0119] (6) Remove the PBST solution, add 30 μL of streptomycin-peroxidase solution to each tissue, incubate at room temperature for 30 min, and wash 3 times with PBST buffer for 5 min each time.

[0120] (7) DAB color development: Use freshly prepared DAB color development solution (1μL:1000μL), incubate at room temperature for 90s, and then stop the color development with pure water.

[0121] (8) Hematoxylin staining: After adding hematoxylin dye and incubating in the room for 4 minutes, rinse slightly with pure water, then differentiate with 1% hydrochloric acid alcohol solution for 20 seconds, rinse slightly with pure water, turn blue with weak ammonia water for 1 minute, rinse slightly with pure water, and place in pure water.

[0122] (9) Dehydration and transparency: Soak the slices in full concentration ethanol for 1 minute, then soak them in xylene for 1 minute to achieve transparency.

[0123] (10) Seal the film with neutral resin liquid and take a picture.

[0124] 1.5.7 DHE-ROS staining experiment

[0125] (1) Select pre-treated thoracic aortic sections and kidney paraffin sections to be stained.

[0126] (2) Wash 3 times with PBS buffer for 5 min each time.

[0127] (3) Stain with DHE staining solution at room temperature in the dark for 45 minutes.

[0128] (4) Wash 3 times with PBS buffer for 5 min each time, and avoid light.

[0129] (5) After mounting with DAPI, observe under a microscope and take pictures.

[0130] 1.5.8 Immunofluorescence assay

[0131] Immunofluorescence staining was performed on frozen sections of mouse thoracic aorta (F4 / 80, CD3, and α-SMA markers). The frozen sections were removed from the -20°C freezer and left at room temperature for 5 minutes.

[0132] (1) Pre-cool the acetone in a -20℃ refrigerator for 30 minutes.

[0133] (2) Fix the slices in acetone for 15 min.

[0134] (3) Let it evaporate at room temperature for 10 minutes until the acetone has evaporated completely.

[0135] (4) Wash 3 times with PBS buffer for 5 min each time.

[0136] (5) Add 50 μL of primary antibody solution, let stand at room temperature for 1 hour, and then freeze overnight at 4°C.

[0137] (6) Wash 3 times with PBST buffer for 5 min each time.

[0138] (7) Add 50 μL of the corresponding secondary antibody and incubate at room temperature for 1 hour, taking care to avoid light.

[0139] (8) Wash 3 times with PBST buffer for 5 min each time.

[0140] (9) After mounting with DAPI, observe under a microscope and take pictures.

[0141] 1.6 In vitro microvascular activity assay (vascular endothelial function assay)

[0142] 1.6.1 Experimental Preparation

[0143] The night before the experiment, prepare the required 5% CO2 / 95% O2 mixed gas, Physiological Saline Solution (PSS) solution, High potassium Physiological Saline Solution (KPSS) solution, and L-phenylephrine (10... -3 mol / L), acetylcholine (Ach) (10 -1 mol / L), sodium nitroprusside SNP (10 -1 Prepare a solution of 1 mol / L acetic acid, 8% acetic acid, and 95% ethanol, and store it in a refrigerator at 4°C.

[0144] Table 2 PSS solution preparation

[0145]

[0146]

[0147] (1) Prepare 1L of PSS solution

[0148] a. First, weigh 1.109 g of CaCl2 and dissolve it in 10 mL of pure water. After it is fully dissolved, filter it through a 0.22 μm filter and store it in a refrigerator at 4 °C.

[0149] b. Add all the substances except the 1 mol / L CaCl2 solution to 800 mL of pure water in the order shown in Table 3, and stir thoroughly to dissolve.

[0150] c. While stirring, add 1.6 mL of 1 mol / L CaCl2 solution and stir thoroughly to dissolve.

[0151] d. Add the remaining 200mL of pure water, stir thoroughly, measure the pH value with a pH meter, correct the pH value to 7.4, and store in a refrigerator at 4℃.

[0152] Table 3. Preparation of KPSS Solution

[0153]

[0154] (2) The preparation method of KPSS solution is the same as that of PSS solution, but the reagent dosage is different, as shown in Table 3.

[0155] (3) L-phenylephrine (10) -3 mol / L), acetylcholine (Ach) (10 -1 mol / L), sodium nitroprusside SNP (10 -1 Weigh out the corresponding mass of each (mol / L), dissolve and mix with pure water, and store at 4℃.

[0156] 1.6.2 Experimental Procedure:

[0157] (1) Separation and fixation of the aorta

[0158] a. Aerate and oxygenate both bottles of PSS for 30 minutes in advance. Preheat one bottle of oxygenated PSS to 37°C and keep the other bottle at room temperature. Preheat the KPSS in the same way.

[0159] b. Under a stereomicroscope, carefully separate the intact blood vessels and transfer them to a culture dish for fixation (do not pull on the blood vessels throughout the process). Use oxygenated room temperature PSS in the culture dish.

[0160] c. After removing the connective tissue and adipose tissue from the abdominal aortic segment using ophthalmic scissors and micro forceps, cut out a vascular ring about 2 mm long (without breaking the blood vessel). First, thread a 2 cm long steel wire through the ring in a petri dish for later use.

[0161] d. Add 5 mL of room temperature PSS to the chamber, fix the blood vessel with the wire threaded on to one end of the sensor (the sensor is very sensitive, do not damage it by force), tighten the wire with a screwdriver (the wire tightening direction is the same as the screw tightening direction), then pass another wire through the blood vessel and fix it to the end of the spiral ruler (note that the two wires should not cross), and ensure that the wires are straight and tight (the two wires are kept on the same horizontal plane).

[0162] e. After the blood vessel is fixed, check the position of the two steel wires and adjust them so that the inner wall of the blood vessel rests on the steel wires.

[0163] f. Place the chambers with fixed blood vessels onto the interface, connect the sensor cable, close the bath lid, turn on the ventilation, and heat. After the blood vessels of all four chambers are fixed, change the solution and raise the temperature to 37°C.

[0164] g. The tension value is zeroed after the temperature rises to 37°C.

[0165] h. After zeroing, replace the preheated PSS buffer solution at 37°C every 20 minutes. During the first equilibration, slowly rotate the screw gauge to allow the tension value to rise slowly until the blood vessel is stretched to the predetermined initial pretension of 2mN (stretch the blood vessel to 2mN within 20 minutes); after adjusting to the predetermined tension, check the initial value every 5 minutes to see if it has decreased, and stretch the vessel promptly if it has decreased; the total equilibration time lasts approximately 60 minutes.

[0166] (2) KPSS solution stimulates vascular activity recovery

[0167] After zeroing the tension, withdraw the solution from the bath and add 5 mL of 60 mM KPSS for stimulation. Once the tension reaches a stable plateau, wait approximately 10-15 minutes, then elute with preheated and oxygenated PSS solution at 37°C until baseline is reached. After reaching baseline, wait approximately 10 minutes. A second stimulation with KPSS solution can be performed, or only one stimulation may be needed, depending on the specific circumstances.

[0168] (3) Endothelium-dependent relaxation test (Ach)

[0169] Add 1 μM shrinkage agent (5 μL 10) -3 Add mol / L L-phenylephrine to 5 mL PSS solution, and after the curve reaches the plateau phase (approximately 10-15 min), perform a diastolic test, adding gradient concentrations (10 mol / L L-phenylephrine to 5 mL PSS solution). -7 -10 -1 Add mol / L Ach to 5 mL.

[0170] The concentration gradient for stimulating endothelial relaxation in PSS is:

[0171] 5μL 10 -7 Add Ach to 5 mL of PSS, final concentration 10. -10 M;

[0172] 4.5μL 10 -6 Add Ach to 5 mL of PSS, final concentration 10. -9 M;

[0173] 4.5μL 10 -5 Add Ach to 5 mL of PSS, resulting in a final concentration of 10. -8 M;

[0174] 4.5μL 10 -4 Add Ach to 5 mL of PSS, final concentration 10. -7 M;

[0175] 4.5μL 10 -3 Add Ach to 5 mL of PSS, final concentration 10. -6 M;

[0176] 4.5μL 10 -2 Add Ach to 5 mL of PSS, resulting in a final concentration of 10. -5 M;

[0177] 4.5μL10 -1 Add Ach to 5 mL of PSS, resulting in a final concentration of 10. -4 M;

[0178] 45μL 10 -1 Add Ach to 5 mL of PSS, resulting in a final concentration of 10.-3 M.

[0179] The measured values ​​and 10 -10 Compare the M Ach values.

[0180] (4) Endothelial-independent vasodilation test

[0181] After eluting to baseline with PSS solution, stimulate once with KPSS solution, then elute to baseline with PSS solution again, and add 1 μM systolic inhibitor (5 μL L-phenylephrine 10). -3 Add M to 5 mL PSS, and wait for the curve to reach the plateau phase (approximately 10-15 min) before performing a diastolic test; do not wash out blood vessels, add gradient concentrations (10 mL PSS to 5 ... -8 -10 -2 1 mol / L SNP was added to 5 mL of PSS to stimulate endothelial relaxation, with the following concentration gradient:

[0182] 5μL 10 -8 SNP to 5 mL PSS, final concentration 10. -11 M

[0183] 4.5μL 10 -7 SNP to 5 mL PSS, final concentration 10. -10 M

[0184] 4.5μL 10 -6 SNP to 5 mL PSS, final concentration 10. -9 M;

[0185] 4.5μL 10 -5 SNP to 5 mL PSS, final concentration 10. -8 M;

[0186] 4.5μL 10 -4 SNP to 5 mL PSS, final concentration 10. -7 M;

[0187] 4.5μL 10 -3 SNP to 5 mL PSS, final concentration 10. -6 M;

[0188] 4.5μL 10 -2 SNP to 5 mL PSS, final concentration 10. -5 M.

[0189] 1.7 Determination of FFA content

[0190] (1) Solvent preparation

[0191] Reagent 1: Prepare according to the ratio of n-heptane: anhydrous methanol: chloroform = 24:1:25, tighten the cap and mix well, store at 2-8℃, and seal promptly after use;

[0192] Reagent 2: Pour Reagent 2B into Reagent 2A and heat and shake at 40℃ for 20 minutes to dissolve. It can be stored at 2-8℃ for 3 months. Before using Reagent 2, preheat it at 37℃ for more than 20 minutes.

[0193] Reagent 3: Before use, take one bottle and add 13mL of anhydrous ethanol to dissolve it completely. It can be stored at 2-8℃ for 2 weeks.

[0194] (2) Preparation and dilution of standard: Before use, transfer the reagent to a 10mL glass bottle, add 7.8mL of chloroform to dissolve it completely, which is a 5μmol / mL palmitic acid standard solution. Store the unused reagent at 2-8℃ for 4 weeks. Dilute the standard with chloroform to 1, 0.8, 0.6, 0.4, 0.2, 0.1 and 0.05μmol / mL.

[0195] (3) Add 30 μL of distilled water to the control tube, 30 μL of chloroform to the blank tube, and 30 μL of serum to the test tube. Add 300 μL of reagent one and 120 μL of reagent two to each of the three tubes.

[0196] (4) After shaking for 15 minutes, centrifuge at 3000 rpm for 10 minutes.

[0197] (5) Take 50 μL of the upper layer solution into a new tube and add 200 μL of reagent three.

[0198] (6) Shake well for 5 min, let stand for 15 min, take 0.2 mL into a 96-well plate, measure the absorbance at 550 nm and record it.

[0199] (7) Establish a standard curve and calculate the results.

[0200] 1.8 Determination of 12-HEPE content

[0201] (1) The content of 12-HEPE was determined by liquid chromatography-mass spectrometry.

[0202] Analysis was performed using a UPLC-MS / MS (LC-MS) system consisting of an ACQUITY H-Class UPLC and a XEVOTQS-micro triple quadrupole mass spectrometer. The chromatographic column was a UPLCBEH C2000. 18The chromatographic column (2.1 mm × 50 mm, 1.7 μm) was used, and the column temperature was 25 °C. Chromatographic conditions: the mobile phase was acetonitrile-0.1% formic acid aqueous solution, with gradient elution at a flow rate of 0.4 mL / min. The gradient elution program was as follows: 0–0.3 min, maintain 10% acetonitrile; 0.3–1.5 min, increase the acetonitrile content from 10% to 90%; 1.5–2.6 min, maintain 90% acetonitrile; 2.6–2.7 min, decrease the acetonitrile content from 90% to 10%; 2.7–4.0 min, maintain 10% acetonitrile.

[0203] (2) Mass spectrometry conditions

[0204] The desolvation gas (nitrogen) flow rate was 850 L / h, the cone gas (nitrogen) flow rate was 50 L / h, the capillary voltage was 2.4 kV, the ion source temperature was 145℃, and the desolvation temperature was 450℃. HEPE was detected using electrospray positive ion mode (ESI+) and multiple reaction monitoring (MRM) mode. The ion for quantitative analysis was: m / z 317.36→179.21.

[0205] 1.9 Statistical Analysis

[0206] All experimental data were entered into GraphPad Prism 9.0 statistical software for statistical analysis. The main experimental data came from three repeated experiments and are expressed as mean ± standard error (Mean ± SEM). ANOVA was used to analyze the variability between groups, and t-tests were used for pairwise comparisons. *P < 0.05 was considered statistically significant.

[0207] 2. Experimental Results

[0208] 2.1 Hypertension induces FFA metabolic disorders and reduces 12-HEPE levels

[0209] Clinical results indicate that the circulating FFA level is higher in hypertensive patients than in healthy individuals. Figure 1 (A). Animal results also showed that Ang II induced an increase in circulating FFA levels ( Figure 1 (B). Subsequently, 12-HEPE was screened from lipidomics, and circulating 12-HEPE levels were detected using LC-MS. Clinical and animal studies revealed that 12-HEPE levels decreased during hypertension. Figure 1 (C and D in the middle).

[0210] 2.2 12-HEPE has a blood pressure-lowering effect.

[0211] This invention investigates whether 12-HEPE can improve hypertension. Hypertensive mice were given 12-HEPE, and their blood pressure was monitored. Results showed that the systolic and diastolic blood pressure of the 12-HEPE group were significantly lower than those of the control group. Figure 2 (A and B in the middle).

[0212] 2.3 12-HEPE improves Ang II-induced hypertensive nephropathy

[0213] This invention evaluated the effects of 12-HEPE on hypertension-induced renal lesions. H&E staining showed that 12-HEPE significantly reduced glomerular size ( Figure 3 (A). Sirius red and Masson's staining revealed that the areas of collagen deposition and fibrosis in the kidneys of mice treated with 12-HEPE were reduced. Figure 3 (B and C). DHE detection of reactive oxygen species (ROS) accumulation indicated that the renal oxidative stress in the treatment group mice was significantly lower than that in the hypertensive control group (B and C). Figure 3 (D). 12-HEPE alleviates kidney damage caused by hypertension.

[0214] 2.4 12-HEPE improves AngII-induced hypertension and vascular complications

[0215] This invention evaluated the effects of 12-HEPE on hypertension-induced vascular lesions. H&E staining was used to observe the morphology of the aorta; 12-HEPE significantly reduced aortic wall thickness. Figure 4 (A). Sirius red staining revealed a reduction in aortic collagen deposition in mice treated with 12-HEPE ( ). Figure 4 (B). DHE detection of reactive oxygen species (ROS) generation showed that the aortic oxidative stress in the drug-treated group was significantly lower than that in the hypertensive control group. Figure 4 (C). Subsequently, the expression of aortic α-SMA was examined, and the results showed that 12-HEPE reduced the degree of aortic fibrosis in mice (C). Figure 4 (D).

[0216] 2.5 12-HEPE improves AngII-induced vascular dysfunction

[0217] This experiment investigated the effects of 12-HEPE on isolated blood vessel function. A concentration-dependent vasodilatory vascular ring assay of the abdominal aorta in response to acetylcholine (Ach) or sodium nitroprusside (SNP) showed that AngII-treated WT mouse abdominal aortas exhibited decreased endothelial vasodilation induced by Ach, and impaired endothelial function in hypertensive mice. Endothelial vasodilation in the abdominal aorta of mice treated with 12-HEPE was significantly restored. There was no significant difference in the vasodilatory response of the abdominal aorta to SNP among the different groups. Figure 5 (A and B in the middle).

[0218] 2.6 12-HEPE stimulates FFAR4 expression in the kidneys of hypertensive mice

[0219] This experiment investigated whether 12-HEPE, which improves hypertension, regulates renal FFAR4 expression levels. Western blot results showed that 12-HEPE significantly upregulated renal FFAR4 protein levels. Figure 6 (A and B in the middle).

[0220] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. The application of a 12-hydroxyeicosalicylic acid in the preparation of health foods that help maintain healthy blood pressure levels.

2. A health food product that helps maintain healthy blood pressure levels, characterized in that, The main active ingredient is 12-hydroxyeicosalicylic acid.

3. The health food product as described in claim 2, characterized in that, It also includes food science-acceptable additives.

4. The use of a 12-hydroxyeicosapate acid in the preparation of a drug for the prevention and / or treatment of hypertension.

5. A drug for the prevention and / or treatment of hypertension, characterized in that, The main active ingredient is 12-hydroxyeicosalicylic acid.

6. The drug as described in claim 5, characterized in that, It also includes pharmaceutically acceptable excipients.

7. The use of a 12-hydroxyeicosapate acid in the preparation of a drug for treating complications of hypertension.

8. The application as described in claim 7, characterized in that, The complications of hypertension include hypertensive nephropathy and hypertensive vascular disease.

9. A drug for treating complications of hypertension, characterized in that, The main active ingredient is 12-hydroxyeicosalicylic acid.

10. The medicament as claimed in claim 9, characterized in that, It also includes pharmaceutically acceptable excipients.

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