WO2020010958A1 - Application of metrnl protein or gene in blocked blood vessel disease - Google Patents

Abstract

Disclosed in the present invention is an application of Metrnl in diagnosis of atherosclerosis. A Metrnl-/-ApoE-/-double knockout atherosclerotic rat model and an EC-Metrnl-/-ApoE-/-atherosclerotic rat model are constructed. The experiment shows that the severity of atherosclerosis, the area of atherosclerotic plaque, and the degree of atherosclerosis and inflammatory infiltration in an aortic root of two Metrnl knockout rats are higher than a rat in a control group. The experiment shows that a vascular endothelial cell can secrete Metrnl, and the vascular endothelium and blood Metrin levels decrease during atherosclerosis, and the degree of decrease in Metrnl levels increases as the disease is exacerbated. The antithrombotic effect of a Metrnl recombinant protein does not have a species difference, and represents a dose-effect relationship on the inhibition effect of a clot contraction reaction. By comparing the function and characteristic of the Metrnl protein with a classical thrombolytic drug, it is found that the antithrombotic effect of Metrnl can reduce an adverse reaction of bleeding.

Description

Application of Metrnl protein or gene in vascular obstructive diseases Technical field

The invention relates to the technical field of medicine, in particular to the application of Metrnl protein or gene in vascular obstructive diseases.

Background technique

Vascular occlusion refers to the occlusion of blood vessels due to slow blood flow, changes in blood composition, or increase in blood viscosity due to age, diet, and other factors. Vascular occlusive diseases include: thrombosis of arteries on the basis of sclerosis, arterial embolism after cardiac thrombolysis, venous thrombosis, impeded blood return after varicose veins, etc. The more common include cerebrovascular occlusion, coronary coronary occlusion, etc. Atherosclerosis, diabetes, hyperlipidemia, and hypertension can accelerate their development. The goal of treatment of vascular obstructive diseases is recanalization of blood vessels. Currently commonly used drugs include streptokinase (SK), acyl plasminogen streptokinase activator complex (APSAC), urokinase (UK), and tissue-type plasminogen. Activator (t-PA), etc. However, the existing thrombolytic drugs can cause adverse reactions caused by the reduction of fibrinogen and other coagulation factors, and the clinical manifestations are local bleeding, subcutaneous sheet bleeding, and even intracranial bleeding.

As the common pathological basis of cardiovascular and cerebrovascular diseases, atherosclerosis (AS) has long been one of the focuses and hotspots in the field of cardiovascular disease. AS is also the main cause of disability and death of cardiovascular disease. Therefore, how to prevent and treat AS is very important for the treatment of cardiovascular disease patients and the improvement of long-term prognosis.

Metrnl, also known as Subfatin, Cometin, Interleukin39, is a new type of adipokine discovered by our laboratory (Miao Chaoyu's research group) in recent years. It is highly expressed in subcutaneous adipose tissue, intestinal epithelial tissue and other parts. It is a homologous protein with Meteorin, but Expression distribution and function are quite different. At present, there are few studies on the function of Metrnl protein. Limited studies have shown that Metrnl is functionally active in aspects such as insulin sensitivity, axon growth, white fat browning, and chondrocyte differentiation. International Patent Publication No. WO2010009732A1 and Chinese Patent Publication No. CN102164611A disclose the application of Metrnl in reducing hearing loss in the inner ear. Chinese Patent Publication Nos. CN103536903A, CN103536904A and International Patent Publication No. WO2015062167A8 disclose the application of Metrnl in lowering blood lipids and blood glucose. Chinese Patent Publication No. CN104808004A discloses the application of Metrnl in the diagnosis of colon cancer. However, the use of Metrnl protein or gene in vascular obstructive diseases has not been reported yet.

Summary of the invention

The first object of the present invention is to provide a new therapeutic use of Metrnl protein or gene in view of the shortcomings in the prior art.

In a first aspect, the use of Metrnl proteins or genes or their synergists in the preparation of anti-atherosclerotic drugs is provided.

In a second aspect, the application of Metrnl protein or gene in a drug for treating atherosclerosis is provided.

In a third aspect, there is provided the use of a Metrnl protein or gene or their synergist in the preparation of a medicament, the medicament being used for:

a) Inhibiting the formation of atheromatous plaque and plaque area in the aorta under a high-fat diet; or

b) Inhibiting thickening of aortic atherosclerosis under a high-fat diet; or

c) Reduce the expression of inflammation-related factors VCAM-1, ICAM-1, TNFα, IL-6 and macrophage marker molecules F4 / 80, CD68 in the aorta under high-fat diet.

The medicament can be made into various dosage forms by using conventional methods in the medical field, using Metrnl protein as an active ingredient, and pharmaceutically acceptable excipients. When used for oral administration, it can be prepared into conventional solid preparations such as tablets, powders, or capsules; for injection, it can be prepared into injection solutions. In various preparations, the active ingredient has a weight content of 0.1 to 99.9%, and a preferred weight content is 0.5 to 90%.

The medicament can be applied to individuals in need of treatment through different routes such as intraperitoneal injection, subcutaneous injection, intravenous injection, intramuscular injection, intra-lymph node injection, and mucosal medication. The individual can be a human or an animal. The dosage is generally 0.01 to 1000 mg / kg of body weight / day, which can be specifically changed according to the age and condition of the individual.

A second object of the present invention is to provide a method for treating atherosclerosis.

In order to achieve the above object, the technical solution adopted by the present invention is: a method for treating atherosclerosis, which comprises administering an effective amount of Metrnl protein or a synergist to an individual in need of treatment, Self-agonist, up-regulator or stabilizer.

A third object of the present invention is to provide a new diagnostic application of Metrnl protein.

In a first aspect, the use of Metrnl protein or a specific antibody thereof is provided for preparing a diagnostic reagent or a kit for detecting atherosclerosis.

In a second aspect, the use of Metrnl protein or a specific antibody thereof is provided for preparing a diagnostic reagent or a kit for detecting the degree of atherosclerotic disease.

In a third aspect, the use of Metrnl protein is provided as a diagnostic marker for detecting atherosclerosis.

A fourth object of the present invention is to provide a method for detecting atherosclerosis.

To achieve the above object, the technical solution adopted by the present invention is: a method for detecting atherosclerosis, the method is to detect the content of Metrnl protein in a serum sample, so as to determine whether the subject derived from the serum sample is suffering Atherosclerosis.

In another preferred example, the test is a serum test.

In another preferred example, the serum detection is an ELISA method.

In another preferred example, the detection is to prepare an antibody against Metrnl protein by a conventional method, and establish a qualitative or quantitative method for detecting Metrnl protein. By detecting the content of Metrnl protein in the serum sample, it is determined whether the subject from which the serum sample originates has an artery. Atherosclerosis. The specifics are as follows: first, measure the content of Metrnl protein in the serum sample to be tested, and then compare it with the content of Metrnl protein in the normal human serum sample to determine whether the content of Metrnl protein in the serum sample to be tested is reduced, and judge the test based on the comparison result Whether the subject of the serum sample has atherosclerosis.

A fifth object of the present invention is to provide a method for detecting the degree of disease in patients with atherosclerosis.

To achieve the above object, the technical solution adopted by the present invention is: a method for detecting the degree of disease in atherosclerosis patients, the method is to detect the content of Metrnl protein in a patient's serum sample, thereby determining the origin of the serum sample The degree of atherosclerotic disease in the subject.

In another preferred example, the detection is to prepare an antibody against Metrnl protein by a conventional method, establish a qualitative or quantitative method for detecting Metrnl protein, and continuously measure the content of Metrnl protein in serum samples of atherosclerotic patients at different times, Based on this, the degree of atherosclerotic disease in the subject from which the serum sample was derived was determined. The details are as follows: The Metrnl protein content in serum samples of patients with atherosclerosis is measured for multiple consecutive days and compared. If the Metrnl protein content (concentration) in the patient's serum sample decreases, it indicates that the disease is aggravated. An increase in the content (concentration) of Metrnl protein indicates a reduction in disease severity.

The advantages of the present invention are:

1. The present invention found for the first time that Metrnl can be used to diagnose and treat atherosclerosis.

2, the present invention is constructed Metrnl ^{- / -} ApoE ^{- / -} double knockout mouse model of atherosclerosis and arterial ^{EC-Metrnl - / - ApoE -} / - mouse model of atherosclerotic arteries, experiments show: Metrnl ^{- / -} ApoE ^{- / -} double knockout mice aorta compared with the control mice had more white ^{plaque, EC-Metrnl - / - ApoE} - / - mice than in control mice aorta having more white atheroma Block; Metrnl ^-/- ApoE ^-/- double-knock mice have larger aortic plaque area than control mice, EC-Metrnl ^-/- ApoE ^-/- mice have aortic plaque area smaller than control Mice; atherosclerotic mice, the aortic root vessels have significant pathological thickening and narrowing of the lumen, and the thickness of the tube wall of Metrnl ^-/- ApoE ^-/- double knock mice is more severe than that of control mice, EC -Metrnl ^-/- ApoE ^-/- mice had a thicker wall than the control group; atherosclerotic mice had obvious lipid plaques at the root of the aorta, and Metrnl ^-/- ApoE ^-/- double knockout mice aortic root atherosclerosis occurs movable higher severity ^{mice, EC-Metrnl - / - ApoE} - / - movable severity of atherosclerosis in the aortic root of mice is also higher ^{Murine; EC-Metrnl - / - ApoE} - / - F4 / 80 molecule aortic root of mice was significantly greater than control ^{mice, EC-Metrnl - / - ApoE} - / - mice in the aortic root α-SMA The molecular expression was significantly more than that of control mice; EC-Metrnl ^-/- ApoE ^-/- mouse pro-inflammatory factors VCAM-1, ICAM-1, TNFα, IL-6 and macrophage marker molecule F4 / 80 and CD68 were up-regulated, while the expression levels of anti-inflammatory factors IL-10, Fizz and Ym1 were not significantly changed.

3. It was found that Metrnl can be secreted by vascular endothelial cells, which is achieved through the classic secretion pathway; the level of vascular endothelial and blood Metrnl decreased during atherosclerosis, and the level of Metrnl decreased with the increase of disease. It is suggested that Metrnl is not only useful for anti-atherosclerosis, but also can be used as a diagnostic indicator of disease.

4. The anti-thrombotic effect of Metrnl in mice was found in the early stage. The present invention has found that there is no species difference in the anti-thrombotic effect of Metrnl recombinant protein. The mouse Metrnl recombinant protein can inhibit rat and human platelet clot retraction, and human Metrnl recombinant protein. Can also inhibit mouse and rat platelet clot contraction response. The inhibitory effect of Metrnl recombinant protein on the clot contraction reaction showed a dose-effect relationship, and both human and mouse Metrnl protein showed inhibitory effect at low concentration (0.1 μg / ml), and the inhibitory effect increased with increasing concentration, of which Metrnl When the protein was 30 μg / ml, the initiation time of clot contraction was more than doubled than normal. Metrnl recombinant protein also inhibited the clot contraction response of platelets in Metrnl gene knockout mice. Metrnl protein can inhibit platelet activation, which is manifested by a decrease in the level of platelet activation marker CD62p. In addition, by comparing the characteristics of anticoagulation between Metrnl protein and the classic thrombolytic drug t-PA, it is found that the differences between the two in the platelet clot contraction test and the whole blood coagulation test, the anticoagulant characteristics of Metrnl can reduce bleeding Adverse reactions.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1: Metrnl ^-/- mouse breeding process and genotype identification.

Figure 2: Metrnl ^-/- ApoE ^-/- double-knock breeding process and genotype identification.

Figure 3: EC-Metrnl ^-/- mouse breeding process and genotype identification.

Figure 4: The cultivation process and genotype identification of EC-Metrnl ^-/- ApoE ^-/- mice.

Figure 5: In vivo morphology of the aorta at 2.5 months of HFD in Metrnl ^-/- ApoE ^-/- double knock mice and their control mice. The black arrow in the figure shows the location of the atheromatous plaque.

Figure 6: The aortic morphology of EC-Metrnl ^-/- ApoE ^-/- and its control mice at 5.5 months of HFD. The white arrow in the picture shows the location of the atheromatous plaque.

Figure 7: Results of oil red O staining in longitudinal section of aorta at 2.5 months of HFD in Metrnl ^-/- ApoE ^-/- double knock mice and their control mice. ** P <0.01 vs control mice.

Figure 8: Results of oil red O staining in longitudinal sections of the aorta at 5.5 months of HFD in EC-Metrnl ^-/- ApoE ^-/- and its control mice. Panel A shows the results of male mice, and Panel B shows the results of female mice. * P <0.05 vs control mice.

Figure 9: Representative images of HE staining of aortic roots at 2.5 months of HFD in C57 wild-type mice and Metrnl ^-/- ApoE ^-/- double knock mice and their control mice.

Figure 10: Representative diagram of aortic root HE staining in EC-Metrnl ^-/- ApoE ^-/- and its control mice at 5.5 months of HFD.

Figure 11: Results of oil red O staining on frozen sections of aortic roots at 2.5 months of HFD in C57 wild-type mice and Metrnl ^-/- ApoE ^-/- double knock mice and their control mice. * P <0.05 vs control mice.

Figure 12: Results of oil red O staining of frozen sections of aortic roots at 5.5 months of HFD in EC-Metrnl ^-/- ApoE ^-/- and its control mice. * P <0.05 vs control mice.

Figure 13: Representative diagram of oil red O staining of frozen sections of aorta at 5.5 months of HFD in EC-Metrnl ^-/- ApoE ^-/- and its control mice.

Figure 14: Representative diagram of immunohistochemistry of frozen section F4 / 80 of aortic root in EC-Metrnl ^-/- ApoE ^-/- and its control mice at 5.5 months of HFD.

Figure 15: Representative map of α-SMA immunohistochemistry of frozen sections of aortic roots in EC-Metrnl ^-/- ApoE ^-/- mice and their control mice at 5.5 months of HFD.

Figure 16: Quantitative detection results of aortic inflammatory factor mRNA in EC-Metrnl ^-/- ApoE ^-/- and its control mice at 5.5 months of HFD. * P <0.05, ** P <0.01 vs control mice.

Figure 17: Metrnl concentration in mouse primary endothelial cell incubation solution and its concentrate.

Figure 18: Concentration of Metrnl in human umbilical vein endothelial cells treated with brefeldin A or eeyarestatin1. ** P <0.01 vs Vehicle Group.

Figure 19: Blood Metrnl levels of EC-Metrnl ^-/- mice. N = 16-17 in each group. ** P <0.01 vs control mice.

Figure 20: Aortic and carotid vascular morphology of mice with different degrees of AS disease.

Figure 21: Blood Metrnl levels in mice with different degrees of AS disease. N = 4-5 in each group, * P <0.05 vs C57 normal mice, ^# P <0.05 vs ApoE ^-/- mice.

Figure 22: Metrnl and CD31 immunofluorescence staining in AS plaque area and non-AS plaque area of mouse aorta.

Figure 23: Blood Metrnl levels in patients with atherosclerosis complicated with myocardial infarction (AS + MI) are significantly lower than those in the control group. There were 11 patients in the control group (8 males and 3 females), and 21 patients in the AS + MI group (17 males and 4 females). *** P <0.001 vs control patients.

Figure 24: There is no species difference in the effect of Metrnl in inhibiting platelet clot contraction. A. Effect of mouse Metrnl recombinant protein on rat platelet clot shrinkage; B. effect of mouse Metrnl recombinant protein on human platelet clot shrinkage; C. human Metrnl recombinant protein on mouse platelet clot shrinkage Effect; D. Effect of human Metrnl recombinant protein on contractile response of rat platelet clots.

Figure 25: Dose-effect relationship of Metrnl recombinant protein inhibiting platelet clot shrinkage response.

Figure 26: Inhibition effect of Metrnl recombinant protein on platelet clot shrinkage response in Metrnl knockout mice.

Figure 27: Metrnl recombinant protein can reduce platelet activation index CD62p content. Each group was n = 3, * P <0.05 vs control group.

Figure 28: Comparison of anticoagulant characteristics of human Metrnl recombinant protein and t-PA recombinant protein. A. Metrnl and t-PA anti-platelet clot contraction experiment; B. Metrnl and t-PA anti-whole blood coagulation experiment; C. Different concentration t-PA anti-whole blood coagulation experiment; D. Effect of Metrnl on whole blood coagulation time , Control group n = 6, each experimental group n = 3, * P <0.05 vs control group; Et-PA had no significant effect on whole blood coagulation time, control group n = 7, and each experimental group n = 3.

detailed description

The specific embodiments provided by the present invention will be described in detail below with reference to the drawings.

Metrnl protein and gene

Herein, the Metrnl protein used may be naturally occurring, for example, it may be isolated and purified from mammals.

In addition, the Metrnl protein may also be artificially prepared, for example, according to conventional genetic engineering techniques. Any suitable Metrnl protein may be suitable for use in the present invention. The Metrnl protein includes a full-length Metrnl protein or a biologically active fragment thereof. As a specific embodiment, the Metrnl protein may be a human Metrnl protein with a size of about 30 kDa, and the amino acid sequence is shown in NP_001004431.1. Metrnl proteins such as rats and mice may also be used.

The amino acid sequence of Metrnl protein formed by substitution, deletion or addition of one or more amino acid residues is also included in the present invention. The Metrnl protein or a biologically active fragment thereof includes a part of a conservative amino acid substitution sequence which does not affect its activity or retains a part of its activity. Proper substitution of amino acids is a well-known technique in the art that can be easily implemented and ensures that the biological activity of known molecules is not altered. These techniques have taught those skilled in the art that, in general, changing a single amino acid in a non-essential amino acid region of a polypeptide does not alter biological activity.

Any biologically active fragment of Metrnl protein can be used in the present invention. Here, the meaning of the biologically active fragment of the Metrnl protein refers to a polypeptide that can still retain all or part of the function of the full-length Metrnl protein. Generally, the biologically active fragment retains at least 50%, 60% to 99%, or 100% of the activity of the full-length Metrnl protein.

The present invention can also use modified or improved all or part of the amino acid Metrnl protein, for example, modified or improved Metrnl protein to promote half-life, effectiveness, metabolism and / or protein efficacy. The modified or modified Metrnl protein may be a conjugate of a Metrnl protein, or a substituted or artificial amino acid thereof. The modified or improved Metrnl protein or gene may have certain differences from the natural Metrnl protein or gene, but it also has the functions described in the present invention and does not cause other adverse reactions or toxicity. That is, any change that does not affect the biological activity of the Metrnl protein or the biological function of the gene can be used in the present invention.

Metrnl synergist and use thereof

The "Metrnl synergist" includes agonists, up-regulators, stabilizers, etc., and refers to any substance that can increase the activity of Metrnl, increase the stability of Metrnl, increase the expression of Metrnl, and increase the effective action time of Metrnl. Substances can be used in the present invention. They can be compounds, small chemical molecules, biomolecules, and the like. The biomolecule may be at a nucleic acid level (including DNA, RNA), at a protein level, or may be a virus product that up-regulates the expression of Metrnl.

Example 1: Construction of a atherosclerotic model of Metrnl genetically engineered mice

1.1 Construction of the Metrnl ^-/- ApoE ^-/- double knock mouse atherosclerosis model

(1) Breeding and genotyping of Metrnl systemic knockout mice (Metrnl ^-/- )

Metrnl systemic knockout mice (Metrnl ^-/- for short) were obtained using the Cre-LoxP gene knockout system (Diabetes2015; 64: 4011-4022) according to the breeding process of FIG. 1A. Among them, Metrnl ^{loxP / loxP} came from this unit, Ella-Cre mice were purchased from Shanghai Nanfang Model Biotechnology Development Co., Ltd., and C57BL / 6J mice were purchased from Shanghai Xipuer-Bikai Experimental Animal Co., Ltd. Genotype identification method: Ella-Cre gene and Metrnl gene need to be identified. The mouse tail genotype was identified using upstream and downstream primers of the corresponding genes. Ella-Cre gene was positive with a 100bp band; the amplified product of the Metrnl knockout sequence was 289bp, and the amplified product of the Metrnl wild sequence was 150bp. As shown in Figure 1B, Metrnl ^+/- Ella-Cre mice showed three bands of 100bp, 150bp, and 289bp (lane 1); Metrnl ^+/- appeared two bands of 150bp and 289bp (lane 2); Metrnl ^-/- full knock The mouse showed a band of 289 bp (lane 4); the wild control mouse showed a band of 150 bp (lane 3).

(2) Breeding and genotyping of Metrnl ^-/- ApoE ^-/- double knock mice

Metrnl ^-/- ApoE ^-/- double knock mice Metrnl ^-/- mice and ApoE ^-/- mice were obtained by mating and breeding according to the incubation process of Fig. 2A, of which ApoE ^-/- mice were purchased from Shanghai Nanfang Biotechnology Development Co., Ltd. Genotype identification method: Metrnl gene and ApoE gene need to be identified. The mouse tail genotype was identified using upstream and downstream primers of the corresponding genes. The amplified product of Metrnl knockout sequence was 289bp, the amplified product of Metrnl wild sequence was 150bp, the amplified product of ApoE knockout sequence was 245bp, and the amplified product of ApoE wild sequence was 155bp. As shown in FIG. 2B, three bands of 289bp, 150bp, 245bp, and 155bp appeared in Metrnl ^+/- ApoE ^+/- mice (lane 3); two bands of 289bp and 245bp appeared in Metrnl ^-/- ApoE ^-/- double-click mice (Lane 6); Wild control mice with double knock mice showed two bands of 150 bp and 245 bp (lane 2).

(3) Preparation of Metrnl ^-/- ApoE ^-/- double knock mouse atherosclerosis model using western high-fat diet

Western high-fat diet feed was purchased from Shanghai Pluton Biotechnology Co., Ltd. The feed formula was 100g of feed: 60g of common breeding feed, 16.4g of lard, 10g of sucrose, 8.5g of casein, 1.3g of cholesterol, and 0.3g of bile salt. , Premix 1.6g, Maltodextrin 1.9g. From 2-3 months of age, mice began to replace their normal diet with high-fat diet (HFD), and examined atherosclerotic indicators at 2.5 months of HFD.

1.2 Construction of EC-Metrnl ^-/- ApoE ^-/- mouse atherosclerosis model

(1) Breeding and genotyping of Metrnl endothelial cell-specific knockout mice (EC-Metrnl ^-/- )

Metrnl endothelial cell-specific knockout mice (ie, Metrnl ^{loxP / loxP} Tek-Cre mice, referred to as EC-Metrnl ^-/- ) were obtained by mating and breeding using the Cre-LoxP gene knockout system according to FIG. 3A incubation process, where Tek- Cre mice were purchased from Shanghai Nanfang Model Biotechnology Development Co., Ltd. Genotype identification method: Tek-Cre gene and Metrnl-flox gene need to be identified. Using the upstream and downstream primers of the corresponding genes to identify rat tail genotypes, a 100bp band was positive for the Tek-Cre gene; the amplified product of the Metrnl-flox sequence was 243bp, and the corresponding amplified product of the wild sequence was 131bp. As shown in FIG. 3B, 100 bp, 243 bp, and 131 bp appeared in Metrnl ^{loxP / wt} Tek-Cre mice (lane 1); 100 bp and 243 bp appeared in EC-Metrnl ^-/- mice (lane 2); the control of special knockout mice was small The mouse appeared 243 bp (lane 3).

(2) Breeding and genotyping of EC-Metrnl ^-/- ApoE ^-/- mice

EC-Metrnl ^-/- ApoE ^-/- double knock mice were obtained by mating and breeding according to the incubation flow of Figure 4A using EC-Metrnl ^-/- mice and ApoE ^-/- mice. Genotype identification method: Tek-Cre needs to be identified Gene, Metrnl-flox gene and ApoE gene. Using the upstream and downstream primers of the corresponding genes to identify rat tail genotypes, a 100bp band was positive for the Tek-Cre gene; the Metrnl-flox sequence amplification product was 243bp, and the corresponding wild sequence amplification product was 131bp; the ApoE knockout sequence amplification product It was 245bp, and the amplified ApoE wild sequence product was 155bp. As shown in FIG. 4B, EC-Metrnl ^-/- ApoE ^-/- double knock mice showed 100 bp, 243 bp, and 245 bp (lane 2), and control mice showed two bands of 243 bp and 245 bp (lane 3).

(3) Preparation of EC-Metrnl ^-/- ApoE ^-/- double knock mouse atherosclerosis model using western high-fat diet feed

Western high-fat diet feeds are the same as above. From 2-3 months of age, mice began to replace their normal ordinary feed with Western high-fat feed, and examined various indicators of atherosclerosis at 5.5 months of HFD.

Example 2: Experimental methods and results of metrnl against atherosclerosis

2.1 Experimental methods

(1) Mouse heart and aorta separation

Mice were intraperitoneally injected with 1% sodium pentobarbital (100mg / kg) for anesthesia. The alcohol and cotton balls were used to moisten their thorax and abdomen. The abdominal cavity and thorax were cut in sequence from below the sternal handle to fully expose the heart and its bottom veins. Blood was taken from the vena cava with a syringe, then a small cut was made in the right atrium of the heart. A 10 ml syringe was filled with pre-chilled 1 × PBS buffer. Then the lungs, liver, stomach, spleen, pancreas, intestine, fat and genital organs were cut off, leaving the heart, aorta and kidneys, and the aorta was separated under a microscope. Take care not to injure the aorta when separating, first remove the fat from each part of the aorta, and then cut the aorta and the body one by one to separate the aorta and the heart connected to it (retain the left and right atrial appendages). Finally, the heart was immersed in 4% paraformaldehyde and fixed; the aorta was fixed in 4% paraformaldehyde or immediately frozen with liquid nitrogen to extract mRNA.

(2) Aortic plaque oil red O staining

Oil red O dye solution preparation method: First use oil isopropyl alcohol to mix oil red O powder (Sigma) into 0.5% oil red O stock solution, filter with filter paper, and use oil red O stock solution and ddH ₂ O according to 3: 2 Mix by volume. Staining method: When the aorta is immersed and fixed in 4% paraformaldehyde for 24 hours, it is transferred to a silicone dish filled with 1 × PBS buffer solution, the aorta is dissected longitudinally, and it is fixed flatly on a silicone surface with a needle. Then discard the PBS buffer, add oil red O staining solution, and let stand for 15 minutes, then drain oil red O staining solution and add 70% ethanol, leave it for 10-15 minutes, then drain 70% ethanol, add ddH ₂ O, Take a picture.

(3) Frozen section of mouse aortic root

After the heart is immersed in 4% paraformaldehyde for 48 hours and fixed, remove it, remove the apical part of the heart along the left and right atrial appendages, and place the root tissue of the heart aorta on the sample surface of the pre-chilled frozen microtome. Tissues were embedded with frozen embedding agent, frozen at -20 ° C for 30 min, and then frozen sections were started. The thickness of each sample was 10 μm when cut to the root of the aorta, and stored at -20 ° C for 30 minutes at room temperature.

(4) HE staining of frozen sections of mouse aortic roots

Remove the frozen section from -20 ° C and bake at 37 ° C for 1h. Place the pieces in order: absolute ethanol × 1min → 95% ethanol × 1min → 85% ethanol × 1min → 75% ethanol × 1min → rinsing under running water × 5min → Drop water drop → hematoxylin × 5min → running water wash × 5min → drop water drop → 0.5% HCl-75% ethanol × 3s → running water wash × 5min → drop water drop → 75% ethanol × 1min → eosin × 5min → absolute ethanol × 1min × 4 times → drying with electric hair dryer → neutral gum sealing sheet.

(5) Oil red O staining of frozen sections of mouse aortic roots

Remove the frozen section from -20 ° C and bake at 37 ° C for 1h. Put the pieces in order: Oil Red O dye solution × 15min → 60% ethanol × 1min → ddH ₂ O × 1min → Drop off the water droplets → Slightly dry → Glycerin Cover film.

(6) Immunohistochemistry of frozen sections of mouse aortic roots

The F4 / 80 antibody was purchased from abcam company, and the α-SMA antibody and SABC kit were purchased from Wuhan Baoshide Biological Engineering Co., Ltd. Immunohistochemical method: Take frozen sections from -20 ° C, bake at 37 ° C for 1h, and draw circles around the tissue of each piece with an immunohistochemical pen. Add 3% H ₂ O ₂ to the circle, and incubate in the dark × 8min → wash with ddH ₂ O × 5min × 3 times → add 0.2% Triton X-100 to the circle, 5min → drop off the water → 1 × PBS buffer wash × 5min × 2 times → Add 5% BSA to the circle, 30min → Add appropriately diluted antibody to the circle, put in a wet box at 4 ° C overnight → 1 × PBS buffer solution wash × 5min × 3 times → drop into the circle Add secondary antibody, 20min → 1 × PBS buffer solution × 5min × 3 times → SABC liquid is added dropwise into the circle, 20min → 1 × PBS buffer solution × 5min × 4 times → 1ml ddH ₂ O to 1.5mL EP tube In the middle, add one drop of ABC reagent to the inside, mix a few drops into the circle after mixing, control the reaction time under the microscope (about 1min) → running water washing × 10min → hematoxylin × 45s → running water washing × 5min → shake off the water droplet → 0.5 % HCl-75% ethanol × 5s → running water rinse × 5min → anhydrous ethanol × 5s → xylene × 5s → slightly dry → neutral gum seal.

(7) Detection of mouse aortic inflammatory factor expression

The kit "RNeasy Micro Kit" for extracting mouse aortic mRNA was purchased from QIAGEN. The method for obtaining and cryopreserving the mouse aorta was the same as the experimental method "(1) Mouse heart and aorta separation". Follow the instructions of the kit to extract aortic mRNA, reverse the mRNA into cDNA at a concentration of 500ng / 10ul, perform QPCR amplification, and use ΔΔCт method for quantitative analysis. The primer sequences used are as follows:

Gene	Forward primer (5 ’→ 3’)	Reverse Primer (5 ’→ 3’)
VCAM-1	TGCCGGCATATACGAGTGTGA	CCCGATGGCAGGTATTACCAAG
ICAM-1	CAATTCACACTGAATGCCAGCTC	CAAGCAGTCCGTCTCGTCCA
TNFα	AAGCCTGTAGCCCACGTCGTA	GGCACCACTAGTTGGTTGTCTTTG

IL-6	CCACTTCACAAGTCGGAGGCTTA	GCAAGTGCATCATCGTTGTTCATAC
IL-1β	TCCAGGATGAGGACATGAGCAC	GAACGTCACACACCAGCAGGTTA
F4 / 80	GAGATTGTGGAAGCATCCGAGAC	GATGACTGTACCCACATGGCTGA
CD68	CATCAGAGCCCGAGTACAGTCTACC	AATTCTGCGCCATGAATGTCC
IL-10	CTTACTGACTGGCATGAGGATCA	GCAGCTCTAGGAGCATGTGG
Fizz1	TTGCAACTGCCTGTGCTTAC	CAAGAAGCAGGGTAAATGGG
Ym1	TTTGATGGCCTCAACCTGGA	AGTGAGTAGCAGCCTTGGAATGTC
GAPDH	GTATGACTCCACTCACGGCAAA	GGTCTCGCTCCTGGAAGATG

2.2 Experimental results

(1) Aortic morphology after mouse aortic atherosclerosis

After the mouse heart was sufficiently perfused with 1 × PBS buffer, the blood in the blood vessels was washed away, and the distribution of aortic plaques could be observed with the naked eye under a low magnification microscope. Figure 5 shows the morphology of aorta in the body of Metrnl ^-/- ApoE ^-/- double knock mice and their control mice at 2.5 months of HFD. The heart, aorta and muscle tissue can be seen. The results show that Metrnl- ^{/ -The} aorta of ApoE ^-/- double knock mice has more white atheromatous plaques (shown by the black arrows in the figure). Figure 6 shows the aortic morphology of EC-Metrnl ^-/- ApoE ^-/- and its control mice at 5.5 months of HFD. The heart and aorta can be seen. The results show that EC-Metrnl ^-/- The aorta of ApoE ^-/- mice has more white atheromatous plaques (shown by yellow arrows in the figure).

(2) Oil red O staining of atherosclerotic plaques in longitudinal sections of mouse aorta

Oil red O is a fat-soluble bright red dye. After oil red O staining, the lipid deposition site is stained red, and the non-lipid deposition site is not colored, so it can reflect the aortic atheromatous plaque. Severity. As shown in Figure 7, in the 2.5-month HFD of Metrnl ^-/- ApoE ^-/- double-click mice, the proportion of the area of the red aortic plaque in the longitudinal section of the entire aorta was significantly higher than that in the control group. As shown in Figure 8, the ratio of the area of atheromatous plaques in the aorta of EC-Metrnl ^-/- ApoE ^-/- mice at 5.5 months of HFD was also higher than that of control mice and male mice. (Figure 8A) and female mice (Figure 8B) were demonstrated.

(3) HE staining results of frozen sections of mouse aortic roots

HE staining is used to observe the morphology of the tissue. Figure 9 shows the results of HE staining of the aortic roots of C57, Metrnl ^-/- ApoE ^-/- double knock mice and their control mice at 2.5 months of HFD. It was found that the vascular wall of the aortic root of C57 wild-type mice without atherosclerosis was thin, while Metrnl ^-/- ApoE ^-/- double-knocking mice and their control mice with atherosclerosis The aortic root vessels had a significant pathological thickening and narrowed lumen, and the thickness of the tube walls of Metrnl ^-/- ApoE ^-/- double-knocked mice was more severe than that of the control group. Figure 10 shows the results of HE staining of aortic roots in EC-Metrnl ^-/- ApoE ^-/- and its control mice at 5.5 months of HFD. Similarly, EC-Metrnl ^-/- ApoE ^-/- mice The tube wall thickened more severely than the control mice.

(4) Oil red O staining results on frozen sections of mouse aorta and aortic root

Oil red O staining of frozen sections of mouse aortic roots reflects the formation of atherosclerotic plaques in the aortic roots. As shown in Figure 11, C57 wild-type mice without atherosclerosis did not have lipid deposits in their aortic roots, but Metrnl ^-/- ApoE ^-/- double knock mice with atherosclerosis and their In control mice, aortic roots had obvious lipid plaques, which caused the old old lumen to narrow to form new ones. The ratio of the area of the red atheroma in the lumen to the area of the old lumen was used to indicate the involvement of the aortic root. Statistics showed that atherosclerosis in the aortic root of Metrnl ^-/- ApoE ^-/- double knock mice Severity was higher than that of control mice. Similarly, as shown in Fig. 12, the severity of arteriosclerosis in the aortic root of EC-Metrnl ^-/- ApoE ^-/- mice was also higher than that in the control group. Figure 13 shows the results of aortic oil red O staining in EC-Metrnl ^-/- ApoE ^-/- mice and their control mice, which is consistent with the aortic root staining results.

(5) Immunohistochemical results of frozen sections of mouse aortic roots

Atherosclerosis is a chronic inflammatory disease. The F4 / 80 molecule is used as a marker of macrophages and can be used to observe the inflammatory response of atherosclerosis. As shown in Figure 14, the expression of F4 / 80 molecules in the aortic roots of EC-Metrnl ^-/- ApoE ^-/- mice at 5.5 months of HFD was significantly higher than that of control mice.

During the development of atherosclerosis, smooth muscle cells migrate from the tunica media to the intima and proliferate. Α-SMA, as a marker of smooth muscle cells, can reflect the severity of atherosclerosis. As shown in FIG. 15, the expression of α-SMA molecules in the aortic root of EC-Metrnl ^-/- ApoE ^-/- mice at 5.5 months of HFD was significantly higher than that of control mice.

(6) Detection of mouse aortic inflammatory factor expression

Atherosclerosis, as a chronic inflammatory disease, changes in the expression levels of many inflammatory factors during its development. As shown in Figure 16, EC-Metrnl ^-/- ApoE ^-/- mice had pro-inflammatory factors VCAM-1, ICAM-1, TNFα, IL-6 and macrophages in aortic tissues at 5.5 months of HFD Cell marker molecules F4 / 80 and CD68 were all up-regulated, IL-1β was up-regulated but not statistically significant, and the expression levels of anti-inflammatory factors IL-10, Fizz and Ym1 were not significantly changed.

Example 3: Endothelial cells can secrete Metrnl, which is achieved through the classical secretion pathway

3.1 Experimental methods

(1) Collection of mouse primary endothelial cell incubation solution and its concentrated solution

When the cells grow to about 90% confluence, the cells are gently rinsed 3 times with 1 × PBS buffer, serum-free endothelial medium is added, and the cells are cultured in a cell incubator for 12 hours. The cell incubation solution was aspirated into a 15 ml centrifuge tube, and centrifuged: room temperature × 1200 rpm × 5 min, and the obtained supernatant was the cell incubation solution. Take 2ml of the original incubation solution into an ultrafiltration centrifuge tube, centrifuge: room temperature × 4000rpm × 20min, the liquid in the concentration tube (about 0.7ml) is the concentrated solution of the incubation solution. Serum-free medium was used as a control treatment group. Store separately at -80 ° C.

(2) Dosing and collection of human umbilical vein endothelial cells

When the cells grew to about 90% confluence, the cells were gently rinsed twice with 1 × PBS buffer. Serum-free medium containing drugs (brefeldin A or eeyarestatin1, each with a final concentration of 5 μg / ml) was added and placed in a cell culture incubator for 24 h. Pipet the incubation solution into a 15 mL centrifuge tube, centrifuge: room temperature × 1200 rpm × 5 min, and suck the supernatant. Serum-free medium was used as a control treatment group. Store separately at -80 ° C.

(3) Determination of Metrnl concentration by ELISA

ELISA kit (# DY6679, R & D system) was used to measure Metrnl levels in mouse primary endothelial cell incubation solution; ELISA kit (# DY7867-05, R & D system) was used to determine human umbilical vein endothelial cell incubation solution Metrnl level. The experiments were performed with double duplicate wells, and the operation steps were strictly performed according to the instructions provided by the kit. The standard curves were highly fit (R ² > 0.99).

3.2 Experimental results

(1) Secretion of endothelial cells Metrnl

As shown in Figure 17, Metrnl was successfully detected in the mouse primary endothelial cell incubation stock solution and its concentrated solution, and the Metrnl concentration in the stock solution was about 1/3 of the concentration in the concentrated solution, which was consistent with the concentration step (2ml The stock solution was concentrated to about 0.7 ml of concentrate). In addition, the concentration of Metrnl in the blank serum-free medium was below the detection limit. This result demonstrates that endothelial cells can indeed secrete Metrnl.

(2) Secretion pathway of endothelial cells Metrnl

Brefeldin A is an inhibitor of Golgi transport function, and eeyarestatin 1 is an inhibitor of endoplasmic reticulum transport channels. As shown in Figure 18, after treating endothelial cells with brefeldin A or eeyarestatin 1, Metrnl was hardly detected in the culture medium, which indicates that the secretion process of endothelial cells Metrnl can be successfully inhibited by brefeldin A and eeyarestatin1, reflecting that the secretion pathway of endothelial cells Metrnl belongs Classical ER-Golgi protein secretion pathway.

(3) Endothelial cell-specific knockout of Metrnl can significantly reduce blood Metrnl levels, with an average decrease of 74%

As shown in Figure 19, the blood Metrnl level of Metrnl endothelial cell-specific knockout mice (EC-Metrnl ^-/- ) was significantly lower than that of normal control mice, with an average decrease of 74%, indicating that endothelial cells are the main blood Metrnl Source of secretion.

Example 4: Endothelial and blood Metrnl levels decrease during atherosclerosis, and Metrnl levels decrease with increasing disease

4.1 Experimental methods

(1) Preparation of a mouse model of atherosclerosis

ApoE ^-/- mice susceptible to atherosclerosis (AS) were purchased from Shanghai Nanfang Model Biotechnology Development Co., Ltd. Preparation of mouse models with different AS levels: Under the same feeding environment, ApoE ^-/- mice were fed with ordinary feed to 7 months of age and randomly divided into two groups. One group continued to be fed with ordinary feed, and the other group was fed with ordinary feed. Feed was changed to Western high-fat diet feed. After 22 weeks, the atherosclerosis of mice (12 months old) was examined.

(2) Isolation of mouse aorta and carotid artery

Isolation of mouse aorta was the same as before. Isolation of the mouse carotid artery was performed after the aortic separation was completed. The mice were supine, cut along the front of the neck, and separated until the common, internal and external carotid arteries were exposed. The carotid arteries near the bifurcation of the blood vessels were taken. .

(3) Mouse aortic immunofluorescence staining

The mouse thoracic aorta was isolated and cut into 2-3mm sections, immersed in 4% paraformaldehyde solution and fixed for at least 48 hours, and frozen sections were made and stored at -20 ° C. Before the immunohistochemistry experiment, the frozen sections were dried at 37 ° C for 2 hours, and then the tablets were rinsed twice with 1 × PBS buffer; the tablets were antigen-recovered using sodium citrate antigen repair solution and microwave heating; immunohistochemistry was used. The pen draws a circle around the tissue; rinses the film 3 times; drops the blocking serum into the circle, and blocks the wet box at room temperature for 2h; shakes off the blocking serum, and drops the diluted Metrnl primary antibody (Abcam, # ab121775) into the circle. , Dilution ratio 1: 200) or CD31 primary antibody (Abcam, # ab121775, dilution ratio 1: 200), and incubated overnight at 4 ° C in a wet box. The next day, the film was rinsed 3 times; the secondary antibody was added dropwise to the circle, and the wet box was incubated at room temperature for 1 hour in the dark; the film was rinsed 3 times; the DAPI dye solution was added to the circle, and the wet box was incubated at room temperature for 3-5 minutes, protected from light. ; Rinse the film 3 times; add anti-fluorescent quencher to the circle, seal the slide, observe and take a picture under a laser confocal microscope.

4.2 Experimental results

(1) Aortic and carotid vascular morphology in mice with different degrees of AS disease

ApoE ^-/- mice are widely used in preclinical studies of AS diseases. They can spontaneously AS under normal dietary conditions, and HFD can accelerate the occurrence and development of AS. As shown in Fig. 20, C57 normal mice without AS disease had clean blood vessels without foreign bodies; ApoE ^-/- mice (12 months old) with spontaneous AS under normal diet had both aorta and carotid arteries. White AS plaques were compared; in contrast, after 22 weeks of HFD in ApoE ^-/- mice (12 months of age), the AS condition was more severe, showing a large number of AS plaques in the aorta and carotid arteries.

(2) Metrnl concentration in blood of mice with different levels of AS disease

Mice with different levels of AS disease (as shown in FIG. 20) were tested for serum Metrnl concentration. As shown in FIG. 21, serum Metrnl levels decreased with the increase of AS disease severity.

(3) Metrnl and CD31 immunofluorescence staining in mouse plaque and non-plaque areas

CD31 molecule is a very abundant endothelial cell-to-cell junction protein, often used as a molecular marker of endothelial cells. Take C57 mice and ApoE ^-/- mice with spontaneous AS under normal diet (as shown in Figure 20), and perform immunofluorescence staining on frozen sections of their thoracic aorta (as shown in Figure 22). CD31 molecules (green (Fluorescence) showed very obvious continuous expression in the vascular wall of normal C57 mice, overlapping with Metrnl staining (red fluorescence); CD31 molecules in non-plaque areas of ApoE ^-/- mice also linearly expressed in the vascular wall, suggesting non-plaque There are relatively complete endothelial cells in the region, and at the same time, the weakened expression of Metrnl in the vascular wall can also be detected. In contrast, the expression of CD31 and Metrnl in the plaque area are very low, almost lacking, indicating that the endothelial cells in the plaque area are damaged. Metrnl is a secreted protein, and this result is not only consistent with the characteristics of exacerbation of vascular endothelium during the development of AS, but also consistent with the above-mentioned decrease in serum Metrnl levels with the increase in AS disease severity.

(4) The blood Metrnl level of patients with AS disease complicated by myocardial infarction also decreased significantly.

Diagnose AS patients with myocardial infarction according to medical records, ECG, biochemical indicators, and vascular imaging. Blood collection, anticoagulation, centrifugation, and plasma measurement are performed. As shown in Figure 23, the blood Metrnl level in AS patients with myocardial infarction is significantly lower than that in the control group. .

The above experiments confirmed that Metrnl can be secreted by vascular endothelial cells, which is achieved through the classic secretion pathway; the level of vascular endothelial and blood Metrnl decreases during atherosclerosis, and the level of Metrnl decreases with the increase of disease. It is suggested that Metrnl is not only useful for anti-atherosclerosis, but also can be used as a diagnostic indicator of disease.

Example 5: Metrnl recombinant protein inhibits platelet clot contraction response without species differences

5.1 Experimental methods

(1) Preparation of platelet suspension

Preparation of mouse platelet suspension: intraperitoneally inject 10% chloral hydrate (300mg / kg) to anesthetize the mice, take a 1ml syringe and wet the inner wall of the syringe with 2% sodium citrate anticoagulant, and reserve 100 μl of anticoagulant in Inside the syringe. After the mouse was numbed, its limbs were fixed, and its abdomen was opened along the midline of the abdomen (to avoid damage to the blood vessels). Carefully poke out the internal organs to expose the abdominal aorta. Use a 1ml syringe with a needle to pierce the abdominal aorta at an angle of 45 ° Blood (about 1ml), immediately after the blood collection, gently invert the syringe, mix the arterial blood and the anticoagulant, and transfer to a 15ml centrifuge tube. Centrifuge (1750rpm × 2min, room temperature), draw the platelet layer into a new 15ml centrifuge tube, continue to add about 300μl physiological saline to the 15ml centrifuge tube containing the remaining blood cells, gently mix, and centrifuge again (1750rpm × 30s, room temperature). Platelet layer. The two platelet layer solutions were mixed together and centrifuged (2750 rpm × 3min, room temperature) to obtain platelet precipitation. The upper transparent PPP (Poor Platelet Plasma) solution was first transferred to a new 1.5 ml centrifuge tube and incubated at 37 ° C for subsequent experiments. Then continue to add an appropriate amount of 37 ° C TB (Tyrode's Buffer) solution (containing 0.04U / ml Apyrase) to a 15ml centrifuge tube, and carefully resuspend the platelets by pipetting to obtain a platelet suspension for subsequent experiments.

Preparation of rat platelet suspension: intraperitoneally inject 10% chloral hydrate (350mg / kg) to anaesthetize the rats, take a 10ml syringe and wet the inner wall of the syringe with 3.8% sodium citrate anticoagulant, and reserve 1ml of anticoagulant in Inside the syringe. After the rat is anesthetized, place it in the supine position, open its abdomen along the midline of the abdomen (to avoid damaging blood vessels), carefully open the internal organs, and expose the abdominal aorta. Use a 10ml syringe with a needle to pierce the abdominal aorta at an angle of 45 ° Blood (about 10ml), immediately after taking the blood, gently invert the syringe, mix the blood with the anticoagulant, and transfer to a 15ml centrifuge tube. Centrifuge (1750rpm × 10min, room temperature), draw the platelet layer to a new 15ml centrifuge tube, centrifuge (2750rpm × 5min, room temperature) to obtain platelet precipitation, first transfer the upper transparent PPP solution to a new 1.5ml centrifuge tube at 37 ° C Incubate for subsequent experiments, and then continue to add an appropriate amount of 37 ° C TB solution (containing 0.04U / ml Apyrase) to a 15ml centrifuge tube, and carefully resuspend the platelets to obtain platelet suspensions for subsequent experiments.

Human platelet suspension preparation: Volunteer elbow vein blood was collected by a hospital nurse, collected into a vacuum blood collection tube with ACD anticoagulant, and the blood was immediately mixed with the anticoagulant and transferred to a 15 ml centrifuge tube. Centrifuge (1750 rpm × 4min, room temperature), aspirate the platelet layer to a new 15ml centrifuge tube, centrifuge (2550rpm × 2.5min, room temperature) to obtain platelet precipitation, first transfer the upper transparent PPP solution to a new 1.5ml centrifuge tube at 37 Incubate at ℃ for subsequent experiments, and then continue to add an appropriate amount of 37 ° C TB solution (containing 0.04U / ml Apyrase) to a 15ml centrifuge tube, and carefully resuspend the platelets by pipetting to obtain platelet suspensions for subsequent experiments.

(2) Drug incubation

Metrnl recombinant protein solution or an equal volume of a control solvent was added to the platelet suspension (250 μl), and the mixture was incubated at 37 ° C. for 15 minutes for experiments. Both human and mouse Metrnl recombinant proteins were purchased from R & D Systems, and the final concentration after adding to platelet suspension was 1.923 μg / ml.

(3) clot shrinkage test

10 μl of autologous PPP solution, 2.5 μl of 100 mM CaCl ₂ solution, and 1 μl of 10 U / ml thrombin were added to the platelet suspension in order, mixed gently, transferred to a transparent glass tube, and the suspension was allowed to stand to observe the shrinkage of the suspension. Real-time photographing was performed.

5.2 Experimental results

As shown in Figure 24, mouse Metrnl recombinant protein can significantly inhibit platelet clot contraction speed in rats (Figure 24A) and human (Figure 24B); similarly, human Metrnl recombinant protein can also significantly inhibit mice (Figure 24C) and Platelet clot contraction rate in rats (Figure 24D). This shows that there is no species difference in the inhibitory effect of Metrnl recombinant protein on platelet clot shrinkage response. The experiment was repeated three times.

Example 6: Metrnl Recombinant Protein Inhibits Platelet Clot Shrinkage Response in a Dose-Effect Relationship

6.1 Experimental method

(1) Preparation of platelet suspension

Mouse platelet suspensions were prepared as before.

(2) Drug incubation

To the platelet suspension (250 μl), equal volumes of different concentrations of Metrnl recombinant protein or a control solvent were added, and the mixture was incubated at 37 ° C. for 15 minutes for experiments. Human and mouse Metrnl recombinant proteins were purchased from R & D Systems, and the final concentrations after being added to the platelet suspension were 0 μg / ml, 0.1 μg / ml, 0.3 μg / ml, 1 μg / ml, 3 μg / ml, 10 μg / ml, 30 μg / ml.

(3) clot shrinkage experiment

Cit.

6.2 Experimental results

As shown in Figure 25, low-dose (0.1 μg / ml) mice Metrnl recombinant protein can inhibit the platelet clot shrinkage response, and this inhibitory effect increases with the concentration of Metrnl (0.1-10 μg / ml) And enhanced. Continue to increase the concentration of Metrnl, and this inhibitory effect continues to increase. Among them, platelets can be observed at 30μg / ml without obvious clot contraction response at 25min. The response time of this group is later than that of the 10μg / ml group, and is shorter than that of the Vehicle group. The start-up time is delayed more than 1 time, and the contraction response speed is reduced. In addition, human Metrnl recombinant protein also showed a similar dose-effect relationship. The experiment was repeated twice each.

Example 7: Metrnl recombinant protein has an inhibitory effect on platelet clot shrinkage in Metrnl knockout mice

7.1 Experimental method

(1) Preparation of platelet suspension

Mouse platelet suspensions were prepared as before.

(2) Drug incubation

Metrnl recombinant protein solution or an equal volume of a control solvent was added to the platelet suspension (250 μl), and the mixture was incubated at 37 ° C. for 15 minutes for experiments. Both human and mouse Metrnl recombinant proteins were purchased from R & D Systems, and the final concentration after adding to platelet suspension was 1.923 μg / ml.

(3) clot shrinkage experiment

Cit.

7.2 Experimental results

As shown in FIG. 26A, the platelet clot contraction response speed of Metrnl fully knocked mice (Metrnl ^-/- , KO) was significantly faster than that of wild control mice (WT), which was consistent with the previous observation results. However, after the addition of Metrnl recombinant protein, the platelet contraction speed of KO mice was significantly reduced, which was comparable to that of the wild control group. Similarly, as shown in FIG. 26B, the platelet clot contraction response speed of Metrnl endothelial cell-specific knockout mice (EC-Metrnl ^-/- ) is also faster than that of control mice (WT). After adding Metrnl recombinant protein, The platelet contraction speed of EC-Metrnl ^-/- mice was significantly slower, even slower than that of the wild control group. This indicates that Metrnl recombinant protein can inhibit the platelet clot contraction response of Metrnl gene knockout mice. The experiment was repeated 3 times each.

Example 8: Metrnl Recombinant Protein Can Inhibit Platelet Activation

8.1 Experimental methods

(1) Preparation of Platelet Rich Plasma (PRP)

The mice were anesthetized by intraperitoneal injection of 10% chloral hydrate (300 mg / kg). A 1 ml syringe was used to wet the inner wall of the syringe with 2% sodium citrate anticoagulant, while leaving 100 μl of the anticoagulant in the syringe. After the mouse was anesthetized, place it in the supine position, fully open the chest cavity, expose the heart and remove the pericardium. Use a 1ml syringe to slowly draw blood (about 1ml) from the confluence of veins at the bottom of the heart. Immediately after taking the blood, reverse the syringe gently. The blood and anticoagulant were mixed and transferred to a 15 ml centrifuge tube. Centrifuge (2000 rpm x 8 min, room temperature), pipette the upper PRP solution into a new 1.5 ml centrifuge tube for subsequent experiments.

(2) Drug incubation

PRP is divided into 4 equal volumes. Add equal volumes of human Metrnl recombinant protein or control solvent at different concentrations, mix and incubate at 37 ° C for 15-20 minutes, and then add 1.5 μl 1U / ml thrombin, mix, and mix. Continue to incubate at 37 ° C for 5 min for subsequent experiments. The final Metrnl concentrations after addition to PRP were 10 μg / ml, 30 μg / ml, and 100 μg / ml, respectively.

(3) ELISA to detect CD62p content

The ELISA kit (Shanghai Haring Biotechnology Co., Ltd., # Hl1006) was used to detect the content of CD62p protein in mouse PRP. The experiments were performed in duplicate wells. The operation steps were strictly performed according to the instructions provided in the kit. The standard curves all have a high degree of fit (R ² > 0.98).

8.2 Experimental results

As shown in FIG. 27, human Metrnl recombinant protein can inhibit platelet activation, and it is shown that under the stimulation condition of thrombin, Metrnl recombinant protein can reduce the concentration of CD62p protein, a platelet activation index in PRP. This experiment consists of 3 dose groups, with 3 cases in each dose group, and the experiment was repeated twice.

Example 9: Comparison of anticoagulant characteristics between Metrnl recombinant protein and t-PA

9.1 Platelet clot contraction test

9.1.1 Experimental method

(1) Preparation of platelet suspension

Mouse platelet suspensions were prepared as before.

(2) Drug incubation

Add human Metrnl recombinant protein, t-PA recombinant protein or an equal volume of control solvent to the platelet suspension, mix and incubate at 37 ° C for 15 min for experiment. Human Metrnl recombinant protein was purchased from R & D Systems, and t-PA recombinant protein was purchased from novoprotein. The final protein concentration after added to the platelet suspension was 1.923 μg / ml.

(3) blood clot contraction test

Cit.

9.1.2 Experimental results

As shown in FIG. 28A, the inhibitory effects of the human Metrnl recombinant protein and the t-PA recombinant protein on the platelet clot shrinkage response in mice were different. Among them, Metrnl recombinant protein can inhibit the speed of platelet clot shrinkage reaction; while the platelet suspension under t-PA does not shrink into a clot but flocculent, that is, no obvious clot shrinkage reaction. The experiment was repeated three times.

9.2 Determination of whole blood coagulation time (test tube method)

9.2.1 Experimental method

(1) Drug preparation

To a clean glass test tube (with an inner diameter of 10 mm), an equal volume of 10 μg / ml, 20 μg / ml human Metrnl recombinant protein, 10 μg / ml t-PA recombinant protein, or a control solvent was added.

(2) Determination of clotting time

The mice were anesthetized by intraperitoneal injection of 10% chloral hydrate (300mg / kg). After the mice were anesthetized, they were placed in the supine position, the chest cavity was fully opened, the heart was exposed and the pericardium was removed. A 1ml syringe was used to confluence the veins at the bottom of the heart. Blood was taken and the time taken for recording the blood was recorded as T _{1. After the} blood was taken, the blood was immediately transferred to a glass test tube pre-medicated, incubated at 37 ° C, and the timing was started immediately. When the time was 3 minutes, the glasses were tilted. Test tube (45 °), observe whether the blood is coagulated, and then tilt the test tube every 30s until the blood does not flow when the test tube is poured. The time when the blood was not observed for the first time (ie, the clotting time) was recorded as T ₂ .

9.2.2 Experimental results

As shown in Figure 28B-E, human Metrnl recombinant protein and t-PA recombinant protein have different characteristics against mouse whole blood coagulation: Metrnl recombinant protein can extend the whole blood coagulation time, but once the blood coagulates, it will no longer lyse ( The longest observation time is 60min); while t-PA recombinant protein has no significant effect on the whole blood coagulation time, but it can be dissolved after the blood coagulation and maintained in a flowing state (the longest observation time is 90min). The experiment was repeated three times.

The above experiments confirmed that there is no species difference in the antithrombotic effect of Metrnl recombinant protein. Mouse Metrnl recombinant protein can inhibit clot retraction in rats and human platelets. Human Metrnl recombinant protein can also inhibit platelet clots in mice and rats. Shrink response. The inhibitory effect of Metrnl recombinant protein on clot contraction showed a dose-effect relationship, and the inhibitory effect of Metrnl protein at low concentration (0.1 μg / ml) appeared in both human and mouse, and the inhibitory effect increased with increasing concentration, of which Metrnl protein 30 μg At ml / ml, the initiation time of clot contraction was more than doubled than normal. Metrnl recombinant protein also inhibited the clot contraction response of platelets in Metrnl gene knockout mice. Metrnl protein can inhibit platelet activation, which is manifested by a decrease in the level of platelet activation marker CD62p. In addition, by comparing the characteristics of anticoagulation between Metrnl protein and the classic thrombolytic drug t-PA, it is found that the differences between the two in the platelet clot contraction test and the whole blood coagulation test, the anticoagulant characteristics of Metrnl can reduce bleeding Adverse reactions.

The above is only a preferred embodiment of the present invention. It should be noted that, for those of ordinary skill in the art, without departing from the method of the present invention, several improvements and additions can be made, and these improvements and additions should also be considered as It is the protection scope of the present invention.

Claims (9)

1. Application of Metrnl proteins or genes or their synergists in the preparation of anti-atherosclerotic drugs, said synergists are selected from agonists, up-regulators or stabilizers.
2. Application of Metrnl protein or gene in medicine for treating atherosclerosis.
3. The application of Metrnl proteins or genes or their synergists in the preparation of medicaments is characterized in that the synergists are selected from agonists, up-regulators or stabilizers, and the medicaments are used for:

   a) Inhibiting the formation of atheromatous plaque and plaque area in the aorta under a high-fat diet; or

   b) Inhibiting thickening of aortic atherosclerosis under a high-fat diet; or

   c) Reduce the expression of inflammation-related factors VCAM-1, ICAM-1, TNFα, IL-6 and macrophage marker molecules F4 / 80, CD68 in the aorta under high-fat diet.
4. A method for treating atherosclerosis, characterized in that it comprises administering an effective amount of Metrnl protein or a synergist to an individual in need of treatment, the synergist is selected from an agonist, an upregulator or a stabilizer .
5. The use of Metrnl protein or a specific antibody thereof is characterized in that it is used for preparing a diagnostic reagent or a kit for detecting atherosclerosis.
6. The use of Metrnl protein or a specific antibody thereof is characterized in that it is used for preparing a diagnostic reagent or a kit for detecting the degree of atherosclerotic disease.
7. The use of Metrnl protein is characterized in that it is used as a diagnostic marker for detecting atherosclerosis.
8. A method for detecting atherosclerosis, characterized in that the method is to detect the content of Metrnl protein in a serum sample, so as to determine whether the subject from which the serum sample is derived has atherosclerosis.
9. A method for detecting the degree of disease in atherosclerotic patients, characterized in that the method is to detect the content of Metrnl protein in a patient's serum sample, thereby determining the degree of atherosclerotic disease in the subject from which the serum sample is derived .

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