CN111135303A

CN111135303A - Application of substance for inhibiting angiopoietin-like protein8

Info

Publication number: CN111135303A
Application number: CN202010055576.3A
Authority: CN
Inventors: 秦彦文; 杨云云
Original assignee: BEIJING INSTITUTE OF HEART LUNG AND BLOOD VESSEL DISEASES
Current assignee: BEIJING INSTITUTE OF HEART LUNG AND BLOOD VESSEL DISEASES
Priority date: 2020-01-17
Filing date: 2020-01-17
Publication date: 2020-05-12

Abstract

The application provides an application of a substance for inhibiting angiopoietin-like protein8, which comprises the following steps: application of substance inhibiting angiopoietin-like protein8 in preparing product for preventing or treating diseases related to vascular remodeling induced by sleep apnea is provided. The substance inhibiting the angiopoietin-like protein8 is applied to a product for preventing or treating sleep apnea-induced vascular remodeling, and by inhibiting the thickening of the vascular wall, the collagen deposition in the vascular wall cells, the disorder of the elastic fibers of the vascular wall, the increase of the blood pressure and the like induced by chronic intermittent hypoxia in a sleep apnea mode, the pathological change of the vascular wall caused by sleep apnea is effectively prevented or slowed down, diseases related to the sleep apnea-induced vascular remodeling are prevented or treated, the treatment effect of the sleep apnea-induced vascular remodeling can be effectively improved, and the application prospect is good.

Description

Application of substance for inhibiting angiopoietin-like protein8

Technical Field

The application relates to the technical field of biomedicine, in particular to application of a substance for inhibiting angiopoietin-like protein 8.

Background

The phenomenon of apnea, which occurs repeatedly during sleep, is called sleep apnea (OSA), which induces chronic intermittent hypoxia and promotes vascular remodeling.

Any alternation of hypoxia and normoxia, as opposed to continuous hypoxia, is referred to as intermittent hypoxia, where chronic intermittent hypoxia may last days or weeks, or even years. Chronic Intermittent Hypoxia (CIH) is a hallmark of sleep apnea and is a major factor leading to arteriosclerotic vascular remodeling.

Vascular remodeling (vascular remodelling) refers to adaptive changes in vessel internal diameter or both, and vessel wall thickness, with structural changes including increased proliferation, migration and extracellular matrix (ECM) synthesis of vascular smooth muscle cells; the functional change is manifested by reduced compliance and altered reactivity to vasoactive substances.

An early indicator of response to vascular remodeling is carotid intimal thickness (IMT), and existing studies demonstrate a significant increase in carotid intimal thickness in patients with sleep apnea, and that the apnea index (AHI) is independently correlated with carotid intimal thickness. The index of the current sleep apnea index mainly comprises chronic intermittent hypoxia, the sleep apnea can induce the chronic intermittent hypoxia, the chronic intermittent hypoxia in the sleep apnea mode can obviously promote vascular remodeling, and the main pathological manifestations of the vascular remodeling induced by the sleep apnea include smooth muscle cell proliferation and apoptosis imbalance, extracellular matrix degradation, elastic fiber fracture, collagen deposition and the like.

However, the treatment effect on the sleep apnea-induced vascular remodeling is still not ideal at present, and the problem to be solved is still needed.

Disclosure of Invention

In view of the above, the embodiments of the present application provide an application of a substance that inhibits angiopoietin-like protein8, so as to solve the technical defects existing in the prior art.

The application discloses application of a substance inhibiting angiopoietin-like protein8 in preparing a product for preventing or treating diseases related to vascular remodeling induced by sleep apnea. .

Further, the product for preventing or treating the sleep apnea-induced vascular remodeling-related disease comprises a product for preventing or treating a sleep apnea pattern chronic intermittent hypoxia-induced vascular remodeling-related disease.

Further, the preventing or treating sleep apnea-induced vascular remodeling-related diseases includes inhibiting sleep apnea pattern chronic intermittent hypoxia-induced vascular wall thickening, collagen deposition within vascular wall cells, vascular wall elastic fiber disorder, elevated blood pressure, elevated blood lipids, expression of human hypoxia-inducible factors, expression of cell proliferation-related genes, expression of proliferating cell nuclear antigens, proliferation of vascular smooth muscle cells, and any combination thereof.

Further, the substance inhibiting angiopoietin-like protein8 is used in combination with other drugs for preventing or treating sleep apnea-induced vascular remodeling in the product.

Further, the substance inhibiting angiopoietin-like protein8 includes an angiopoietin-like protein8 inhibitor.

Further, the inhibitor of angiopoietin-like protein8 comprises an agent that binds to or interacts with angiopoietin-like protein8 in vivo or in vitro to inhibit the biological function of angiopoietin-like protein 8.

Further, the angiopoietin-like protein8 inhibitor includes any one or combination of angiopoietin-like protein8 antibodies, small molecule angiopoietin-like protein8 antagonists, nucleic acid-based inhibitors of angiopoietin-like protein8 expression or activity, peptide-based molecules that specifically interact with angiopoietin-like protein8, receptor molecules that specifically interact with angiopoietin-like protein8, proteins that comprise ligand binding portions of low density lipoprotein receptors, scaffold molecules that bind angiopoietin-like protein8, fibronectin-based scaffold constructs, other naturally occurring repeat protein-based scaffold molecules, and anti-angiopoietin-like protein8 aptamers.

The application also discloses application of the substance for knocking down or knocking out the angiopoietin-like protein8 expression gene in preparing a product for inhibiting sleep apnea-induced vascular remodeling.

The application of the substance for inhibiting the angiopoietin-like protein8, which is provided by the application, can effectively prevent or slow down pathological changes of the vascular wall induced by sleep apnea and further prevent or treat diseases related to sleep apnea induced vascular remodeling by inhibiting the thickening of the vascular wall, collagen deposition in the vascular wall cells, vascular wall elastic fiber disorder, blood pressure rise, blood lipid rise, expression of human hypoxia induction factors, expression of cell proliferation related genes, expression of proliferation cell nuclear antigens and expression of vascular smooth muscle cells under the chronic intermittent hypoxia induction of the sleep apnea mode, so that the substance for inhibiting the angiopoietin-like protein8 is applied to the products for preventing or treating the sleep apnea induced vascular remodeling, and the treatment effect of the sleep apnea induced vascular remodeling can be effectively improved, the application prospect is good.

Drawings

FIG. 1 is a graph comparing the expression levels of mouse angiopoietin-like protein8 according to an embodiment of the present application;

FIG. 2 is a graph of mouse angiopoietin-like protein8 co-localization staining according to an embodiment of the present application;

FIG. 3 is a graph comparing the wall thickness staining of a mouse blood vessel according to an embodiment of the present application;

FIG. 4 is a graph comparing collagen deposition staining in mice according to one embodiment of the present application;

FIG. 5 is a comparison of the staining of spandex fibers on the vascular wall of a mouse according to an embodiment of the present application;

FIG. 6 is a graph showing a broken line comparison of blood pressure in mice according to an embodiment of the present application;

FIG. 7 is a bar graph comparing blood lipids in mice according to an embodiment of the present application;

FIG. 8 is a graph comparing staining of mouse HIF-1 α according to one embodiment of the present application;

FIG. 9 is a graph comparing the levels of genes associated with cell proliferation in mice according to an embodiment of the present application;

FIG. 10 is a graph comparing the staining of mouse proliferating cell nuclear antigen and smooth muscle cells according to an embodiment of the present application.

Detailed Description

The following description of specific embodiments of the present application refers to the accompanying drawings.

In the present invention, unless otherwise specified, scientific and technical terms used herein have the meanings that are commonly understood by those skilled in the art. Also, the reagents, materials and procedures used herein are those that are widely used in the corresponding fields. Meanwhile, in order to better understand the present invention, the definitions and explanations of related terms are provided below.

Angiopoietin-like protein (Angiopoietin-like protein, ANGPTL): is a family of secreted protein factors which are not only related to angiogenesis, but also closely related to lipid metabolism, glucose metabolism, energy metabolism, insulin sensitivity and the like. The angptl family contains 8 members and is a class of secreted proteins. Except Angiopoietin-like protein 5(Angiopoietin-like protein5, ANGPTL5) which is expressed only in humans, other members of ANGPTL are expressed in both humans and mice.

Angiopoietin-like protein 8(Angiopoietin-like protein8, ANGPTL 8): is one of the members of the ANGPTLs family. ANGPTL8, also known as GM6484, RIFL, Lipasin and Betatrophin, consists of 198 amino acids, has a molecular weight of 22kDa, and is located on chromosome 19p13.2 a. Physiologically, ANGPTL8 is secreted by liver-specific expression in humans, while ANGPTL8 is secreted primarily by the liver and adipose tissue in mice.

Small interfering RNA (Small interfering RNA, siRNA): also known as short interfering RNA (short interfering RNA) or silencing RNA (silencing RNA), is a double stranded RNA of 20 to 25 nucleotides in length that has many different biological uses.

Antisense RNA: refers to RNA that, when complementary to mRNA, inhibits expression of a gene directly involved in the onset of a disease. The antisense RNA seals gene expression, has the characteristics of strong specificity and simple operation, and can be used for treating diseases and serious infectious diseases caused by gene mutation or over expression.

C57BL/6J mice: is one of the mouse strains. The strain has low incidence rate of breast cancer of mice, has eye defect, has the incidence rate of lip crack of 20 percent, has the spontaneous rate of lymphocytic leukemia of 6 percent, has resistance to radiation injury, is sensitive to tubercle bacillus, has certain resistance to mousepox virus, has high interferon yield, is sensitive to pertussis susceptibility factors and high alcoholicity, is commonly used for carcinogenesis research, and is a strain commonly used for research on the aspects of oncology, physiology, genetics and the like.

Immunohistochemistry (immunohistochemistry): the method can also be called immunocytochemistry (immunocytochemistry) or immunohistochemical staining, and is characterized in that an antigen-antibody reaction which is a principle that an antigen is specifically combined with an antibody is applied as a basic principle of immunology, and a color developing agent (fluorescein, enzyme, metal ions and isotopes) for marking the antibody is developed through a chemical reaction to determine the antigens (polypeptide and protein) in tissue cells, and the tissue cells are subjected to positioning, qualitative and relatively quantitative research.

Immunofluorescence (immunofluorescence scientific): the fluorescent pigment which does not affect the activity of the antigen-antibody is marked on the antibody (or antigen), and after being combined with the corresponding antigen (or antibody), the antibody shows a specific fluorescent reaction under a fluorescent microscope.

HIF-1 is a nuclear protein with transcriptional activity and has a fairly broad target gene spectrum, including nearly 100 target genes associated with hypoxia adaptation, inflammation development, tumor growth, etc., among which HIF-1 α is a human hypoxia inducible factor.

Western immunoblot (Western Blot): the method is a method in which a protein is transferred to a membrane and then detected using an antibody. Wherein, the known expression protein can be detected by using a corresponding antibody as a primary antibody, and the expression product of the novel gene can be detected by using the antibody of the fusion part.

Hematoxylin-eosin staining method (hematoxylin-eosin staining): HE staining method is short, and one of the staining methods commonly used in paraffin sectioning technology. The hematoxylin staining solution is alkaline, and mainly makes the chromatin in the cell nucleus and the nucleic acid in the cytoplasm bluish; eosin is an acid dye that primarily reddens components in the cytoplasm and extracellular matrix.

Collagen fiber staining (Masson staining): it is a mixture of two or three anionic dyes, collagen fibers are blue, muscle fibers are red, and one of the staining methods for showing fibers in tissues and inflammatory factors.

Dyeing elastic fibers: elastic dyeing is for short. Spandex is a fiber that forms later in connective tissue formation, and is usually evident only in 4-5 weeks of wound repair. The elastic fibers are firm, small and elastic, and are easily stretched to exert an elastic effect on connective tissues. The elastic fiber is composed of elastin, and is also called yellow fiber when fresh. The fiber branches of the elastic fibers are connected into a net, the refractivity is strong, pink with strong refractivity is formed on an HE (high-intensity intense) dye when the content is high, and the coloration is not generated when the content is low. Therefore, it is not easily distinguished from collagen fibers when the amount is large, and cannot be observed when the amount is small. The dyeing of the elastic fiber is mainly used for observing whether the elastic fiber has pathological changes such as hyperplasia, swelling, fracture, breakage, atrophy or defect.

The embodiment provides application of a substance inhibiting angiopoietin-like protein8 in preparing a product for preventing or treating diseases related to vascular remodeling induced by sleep apnea.

Specifically, the substance inhibiting angiopoietin-like protein8 is a substance such as an antibody, a virus, a compound, a composition, a preparation, a kit and/or an apparatus having an effect of inhibiting angiopoietin-like protein8, wherein the preparation may be an oral preparation, an injection preparation, or the like, or may be a liquid preparation, a lyophilized powder preparation, a tablet, a granule, or the like, which is not limited in this application.

The product for preventing or treating the sleep apnea induced vascular remodeling related diseases comprises one or any combination of a product for preventing the sleep apnea induced vascular remodeling related diseases, a product for treating the sleep apnea induced vascular remodeling related diseases and a product for treating the sleep apnea induced vascular remodeling related diseases. The related diseases are diseases generated in the process of forming the sleep apnea-induced vascular remodeling, or diseases with a certain correlation with the sleep apnea-induced vascular remodeling, such as complications and sequelae caused by the sleep apnea-induced vascular remodeling, and the related diseases are not limited in the application. The product is a medicine, a preparation, a kit and/or an instrument and the like with related prevention and/or treatment effects, the medicine can be a medicine compound, a medicine composition, a medicine formula and the like, and the preparation can be a liquid preparation, a freeze-dried powder preparation, an oral preparation, an injection preparation and the like, and the application is not limited to the above.

The sleep apnea can induce chronic intermittent hypoxia, the chronic intermittent hypoxia in the sleep apnea mode can obviously promote vascular remodeling, and the main pathological manifestations of the vascular remodeling induced by the chronic intermittent hypoxia in the sleep apnea mode comprise smooth muscle cell proliferation and apoptosis imbalance, extracellular matrix degradation, elastic fiber breakage, collagen deposition, blood pressure increase, blood fat increase and the like.

In other words, the method can be used for inhibiting the vascular wall thickening induced by the chronic intermittent hypoxia in the sleep apnea mode, inhibiting the collagen deposition in vascular wall cells induced by the chronic intermittent hypoxia in the sleep apnea mode, inhibiting the vascular wall elastic fiber disorder induced by the chronic intermittent hypoxia in the sleep apnea mode, inhibiting the blood pressure rise induced by the chronic intermittent hypoxia in the sleep apnea mode, inhibiting the blood lipid rise induced by the chronic intermittent hypoxia in the sleep apnea mode, inhibiting the expression of the human hypoxia-inducible factor induced by the chronic intermittent hypoxia in the sleep apnea mode, inhibiting the expression of the cell proliferation related gene induced by the chronic intermittent hypoxia in the sleep apnea mode, inhibiting the expression of the proliferation nuclear antigen induced by the chronic intermittent hypoxia in the sleep apnea mode, and inhibiting the proliferation of vascular smooth muscle cells induced by the chronic intermittent hypoxia in the sleep apnea mode Or several to prevent or treat sleep apnea-induced vascular remodeling.

Other drugs for preventing or treating sleep apnea-induced vascular remodeling as described in this example may be aspirin, thromboxane A₂Etc., which the present application does not limit.

Specifically, the angiopoietin-like protein8 inhibitor is an antibody or antigen-binding fragment thereof that specifically binds to angiopoietin-like protein 8. Angiopoietin-like protein8 can be administered to a patient orally, intravenously, intramuscularly, or subcutaneously.

Specifically, the angiopoietin-like protein8 antibody includes angiopoietin-like protein8 neutralizing antibody, the nucleic acid-based inhibitor of angiopoietin-like protein8 expression or activity includes small interfering RNA (sirna), antisense RNA, etc., the peptide-based molecule specifically interacting with angiopoietin-like protein8 includes peptibody (peptibody), etc., and the scaffold molecule binding to angiopoietin-like protein8 includes ankyrin repeat protein (DARPin), HEAT repeat protein, ARM repeat protein, triangle tetrapeptide repeat proteins (tetratricopeptide repeat proteins), etc.

The embodiment also provides application of the substance inhibiting angiopoietin-like protein8 in preparing a product for inhibiting the formation of sleep apnea-induced vascular remodeling.

Specifically, the product for inhibiting the formation of the sleep apnea-induced vascular remodeling is a medicament, a preparation, a kit, and/or an apparatus and the like for inhibiting the occurrence or development of the sleep apnea-induced vascular remodeling.

The embodiment also provides application of a substance inhibiting the expression of the angiopoietin-like protein8 in preparing a product for inhibiting the formation of sleep apnea-induced vascular remodeling.

Specifically, the substance inhibiting the expression of angiopoietin-like protein8 is an antibody, virus, compound, composition, preparation, kit and/or instrument having the effect of inhibiting the expression of angiopoietin-like protein 8.

The embodiment also provides application of the substance for knocking down or knocking out the angiopoietin-like protein8 expression gene in preparing a product for inhibiting the formation of sleep apnea induced vascular remodeling.

Specifically, the angiopoietin-like protein8 is knocked down and the angiopoietin-like protein8 is knocked out in a mode of inhibiting the angiopoietin-like protein8, and the substance for knocking down or knocking out the expression gene of the angiopoietin-like protein8 is an antibody, a virus, a compound, a composition, a preparation, a kit and/or an instrument and the like with the effect of knocking down or knocking out the expression gene of the angiopoietin-like protein 8.

The present embodiment also provides a method for the prevention and/or treatment of a disease or disorder, wherein the disease or disorder is typically and preferably selected from sleep apnea-induced vascular remodeling and related diseases, preferably from sleep apnea-induced vascular remodeling and other symptoms of related diseases, wherein the method comprises administering a substance inhibiting angiopoietin-like protein8 and/or a product preventing or treating sleep apnea-induced vascular remodeling related diseases as described herein, preferably a pharmaceutical or pharmaceutical composition inhibiting angiopoietin-like protein8, which may be prepared by mixing with appropriately selected and pharmaceutically acceptable excipients, vehicles, adjuvants, additives, surfactants, desiccants or diluents, which are known to those skilled in the art and which may be suitable for oral administration, for the prevention and/or treatment of diseases associated with sleep apnea, and/or for the prevention or treatment of diseases associated with sleep apnea-induced vascular remodeling Parenteral or topical administration. Typically and preferably, the pharmaceutical compositions of the present invention are administered in the form of tablets, capsules, powders, granules, pellets, oral or parenteral solutions, suspensions, suppositories, ointments, creams, lotions, gels, pastes and/or containing liposomes, micelles and/or microspheres and the like.

In one aspect, a therapeutically effective amount of a substance that inhibits angiopoietin-like protein8 and/or a product that prevents or treats sleep apnea-induced vascular remodeling-related diseases can be administered to an animal, preferably a human, in need thereof. In another aspect, a therapeutically effective amount of an angiopoietin-like protein 8-inhibiting substance and/or a product for preventing or treating sleep apnea-induced vascular remodeling-related diseases may be administered to an animal, preferably a human, for which it is reasonably expected that such is in need.

Wherein "therapeutically effective amount" herein refers to an amount sufficient to modulate one or more symptoms of said sleep apnea-induced vascular remodeling and related diseases, preferably 10mg/kg per subcutaneous injection, once to three times daily. The substance inhibiting angiopoietin-like protein8 and/or the product for preventing or treating diseases associated with sleep apnea-induced vascular remodeling may be administered by any route, including oral, intramuscular, subcutaneous, topical, transdermal, intranasal, intravenous, sublingual, or intrarectal administration, and may be used in combination with other drugs, the dosage depending on the route of administration, the severity of the disease, the age and weight of the patient or subject, and other factors generally considered by the attending physician.

The present embodiments also provide an article of manufacture, a container or package containing the above-described product for preventing or treating a sleep apnea-induced vascular remodeling-related disease, and written instructions such as a package insert and instructions for administration.

The above examples are further illustrated below with reference to specific experiments.

40C 57BL mice of 6 weeks of age were selected and divided into a blank control group, an ANGPTL8 suppression group, a CIH group and a CIH + ANGPTL8 suppression group, and 10 mice were selected each group.

Wherein, the mice of the blank control group are fed with common feed for 6 weeks without any treatment.

The ANGPTL 8-inhibited group of mice were fed with normal feed for 6 weeks, and the human angiopoietin-like protein8 neutralizing antibody (REGN3776) was diluted to a concentration of 10mg/kg with sterile phosphate buffer solution and injected subcutaneously once a week with the human angiopoietin-like protein8 neutralizing antibody.

Among them, angiopoietin-like protein8 neutralizing antibody REGN3776 was developed by velocimune technology platform from Regeneron company. REGN3776 has a human immunoglobulin g (igg)4 constant region with a stabilizing mutation in the hinge region (serine to proline at position Genbank No. 108).

Mice in the CIH group were fed with normal diet for 6 weeks and were continuously subjected to chronic intermittent hypoxic treatment in sleep apnea mode (equipment: OxyCyclerA84, BioSpherix, Redfield, NY, USA) for 6 weeks with oxygen concentration cycling from 5% -21% to 5% and cycling once every 180 seconds, with oxygen concentration 5% for 60s, oxygen concentration 21% for 60s and oxygen concentration 5% for 60s again, cycling for 8 hours per day.

Mice from the group of the CIH + ANGPTL 8-inhibited were fed with normal diet for 6 weeks and were continuously subjected to chronic intermittent hypoxia treatment in sleep apnea mode (apparatus: OxyCyclerA84, BioSpherix, Redfield, NY, USA) for 6 weeks with oxygen concentration cycling from 5% -21% to 5% once every 180 seconds with oxygen concentration 5% for 60s, oxygen concentration 21% for 60s and oxygen concentration 5% for 60s, cycling for 8 hours each day, and further human angiopoietin-like protein8 neutralizing antibody (REGN3776) was diluted with sterile phosphate buffer solution to a concentration of 10mg/kg, and the mice from this group were subcutaneously injected once a week with the human angiopoietin-like protein8 neutralizing antibody.

After feeding the four groups of mice for six weeks, respectively collecting vascular tissues of the four groups of mice, anesthetizing the mice, opening chest apex to take blood, cutting off right auricle, perfusing physiological saline into left ventricle, taking out thoracic aorta of the mice, fixing with paraformaldehyde, embedding in paraffin for the next day, and slicing to obtain thoracic aorta slices with thickness of 7 μm.

Taking the thoracic aorta section of mice in a blank control group and a CIH group for immunohistochemical staining, namely taking the thoracic aorta vascular tissue section of each group of mice, airing for 10min at room temperature, fixing for 30min by 4% paraformaldehyde, washing for 3 times by Phosphate Buffer Saline (PBS), incubating for 20min by endogenous peroxidase inhibitor, washing for 3 times by PBS, washing for 5min each time, sealing for 30min by serum, and after sucking the serum, keeping the temperature of primary antibody (ANGPTL8, Ab180915,100 mu l, purchased from Abcam) overnight at 4 ℃. The next day, after air drying at room temperature for 30min, the secondary antibody (rabbit antibody or mouse antibody corresponding to the primary antibody, ZM0003, 100. mu.l, purchased from China fir gold bridge) was incubated for 2 hours, after staining with diaminobenzidine (3, 3' -diaminobenzidine, DAB) staining solution (brown positive), hematoxylin was counterstained for about 30s-1min, washed with water, differentiated with 1% hydrochloric acid alcohol, and washed with tap water back to blue. After washing the slices in water, the slices were sequentially put into 70% ethanol-80% ethanol-90% ethanol-95% ethanol-absolute ethanol I-absolute ethanol II-xylene I-xylene II to be dehydrated and transparent, each reagent was placed for 2min, and finally the slices were air-dried in a fume hood, and then sealed with neutral gum, observed and image-collected with an upright microscope (Nikon, Tokyo, Japan), to obtain fig. 1.

As shown in FIG. 1, FIG. 1-A is a photograph showing immunohistochemical staining of a section of thoracic aorta of mice in a blank control group, FIG. 1-B is a photograph showing immunohistochemical staining of a section of thoracic aorta of mice in a CIH group, and FIG. 1-C is a graph showing a comparison of the expression levels of angiopoietin-like protein8 in the thoracic aorta of the two groups of mice, wherein the horizontal axis represents the group, the vertical axis represents the expression level (%) of angiopoietin-like protein8, and the horizontal line at the top of the bar represents the standard deviation of the activity level of angiopoietin-like protein 8.

As can be seen from fig. 1, the activity level of angiopoietin-like protein8 in the thoracic aorta of mice in CIH group was much higher than that of placebo group, so angiopoietin-like protein8 was increased in the chronic intermittent hypoxia-induced vascular remodeling in sleep apnea pattern.

The thoracic aorta section of the mice of the blank control group and the CIH group is taken for co-localization staining, namely, the section is taken out and dried for 10min at room temperature, then fixed for 30min by 4% paraformaldehyde, washed for 3 times by Phosphate Buffer Saline (PBS), washed for 5min each time, incubated for 20min by endogenous peroxidase inhibitor, washed for 3 times by PBS, washed for 5min each time, serum is sealed for 30min, the primary antibody after the serum is sucked dry is mixed with α -SMA primary antibody 1:1 at 4 ℃ (angiopoietin-like protein8 primary antibody is mixed with α -SMA primary antibody 1:1, diluted by PBS 1:200, ANGPTL8 primary antibody, Ab180915, 0.5ml, purchased from Abcam company, α -SMA primary antibody, ZM0003, 0.5ml purchased from China fir bridge) is dried for 30min at room temperature, and then the corresponding secondary antibody is sealed for one hour, stored for 1h at 4 ℃, and then the image is acquired by an upright microscope (Nikon, Tokyo 2, and the image is observed and obtained.

As shown in FIG. 2, FIG. 2-A is a co-localization staining chart of endothelial cells, angiopoietin-like protein8, and nuclei of mice in the blank control group and the CIH group, wherein the first column is a staining chart of endothelial cell CD31 in thoracic aorta section of mice in the blank control group and the CIH group from top to bottom, wherein the red dotted part represents endothelial cells, the second column is a staining pattern of the angiopoietin-like protein8 in the section of the thoracic aorta of mice in a blank control group and a CIH group from top to bottom, wherein the green dotted part represents the angiopoietin-like protein8, the third column is a cell nucleus DAPI staining pattern in the section of thoracic aorta of mice of a blank control group and a CIH group from top to bottom, the blue dotted part represents the cell nucleus, the fourth column is a colocalization staining pattern of endothelial cells, angiopoietin-like protein8 and cell nucleus of mice of a blank control group and a CIH group from top to bottom, and the angiopoietin-like protein8 is almost not expressed in the endothelial cells.

FIG. 2-B is a co-localized staining pattern of vascular smooth muscle cells, angiopoietin-like protein8, and nuclei of mice in blank control group and CIH group, wherein the first column is a staining pattern of vascular smooth muscle cells VSMC in thoracic aorta section of mice in blank control group and CIH group sequentially from top to bottom, wherein red dotted portion represents vascular smooth muscle cells, the second column is a staining pattern of angiopoietin-like protein8 in thoracic aorta section of mice in blank control group and CIH group sequentially from top to bottom, wherein green dotted portion represents angiopoietin-like protein8, the third column is a staining pattern of DAPI in thoracic aorta section of mice in blank control group and CIH group sequentially from top to bottom, wherein blue dotted portion represents nuclei, the fourth column is a co-localized staining pattern of vascular smooth muscle cells, angiopoietin-like protein8, and nuclei of mice in blank control group, CIH group sequentially from top to bottom, it can be seen that the expression of angiopoietin-like protein8 in mice treated with chronic intermittent hypoxia in sleep apnea mode was significantly increased in vascular smooth muscle cells relative to the placebo group.

Therefore, chronic intermittent hypoxia-induced vascular remodeling in sleep apnea pattern results in a significant increase in angiopoietin-like protein8 expression, with angiopoietin-like protein8 being predominantly expressed in vascular smooth muscle cells.

Respectively carrying out HE staining on thoracic aorta slices of each group of mice, namely blowing the slices by electric blowing or baking the slices until the slices are dissolved in wax; dewaxing in xylene I for 15min, and sucking out the liquid with absorbent paper; dewaxing in xylene II for 15min, and sucking the liquid with absorbent paper; dewaxing in xylene III for 15min, and sucking the liquid with absorbent paper; washing with 100% alcohol for 5min, and drying with absorbent paper; washing with 95% ethanol for 5min, and drying with absorbent paper; washing with 80% ethanol for 5min, and drying with absorbent paper; washing with tap water for 2min, and washing off alcohol; staining with hematoxylin (200ml hematoxylin +1.5ml glacial acetic acid) for 3-5min, and washing with tap water for 1 min; differentiating 2s (the slices turn red from blue) by using hydrochloric acid differentiation solution (400ml of 75% alcohol and 3ml of concentrated hydrochloric acid), and washing with tap water (returning blue) for 5 min; eosin staining for 2-3min, and washing with tap water for 30 s; adding 80% alcohol for 30s, and blotting with absorbent paper; adding 95% alcohol for 3min, and drying with absorbent paper; adding 100% alcohol for 3min, and sucking off the liquid with absorbent paper; soaking in xylene I for 5min, and drying with absorbent paper; adding into xylene II for 5min, and drying with absorbent paper; neutral gum mounting, observed by upright microscope (Nikon, Tokyo, Japan) and image acquisition, gave figure 3.

As shown in FIG. 3, FIG. 3-A is a graph showing the staining of the HE of a section of the thoracic aorta of a mouse in a blank control group, FIG. 3-B is a graph showing the staining of the HE of a section of the thoracic aorta of a mouse in an ANGPTL8 inhibition group, FIG. 3-C is a graph showing the staining of the HE of a section of the thoracic aorta of a mouse in a CIH group, FIG. 3-D is a graph showing the staining of the HE of a section of the thoracic aorta of a mouse in a CIH + ANGPTL8 inhibition group, wherein "400X" in the above-mentioned graphs indicates a magnification of 400 times, and FIG. 3-E is a histogram comparing the thicknesses of the vascular wall thicknesses of the four groups of mice, wherein the horizontal axis indicates the group, the vertical axis indicates the thickness of the vascular wall (μm) of the mouse, the horizontal line at the top of.

As can be seen from fig. 3, ANGPTL8 inhibited mice in the group from having a large difference in vascular wall thickness relative to the placebo group, so inhibition of ANGPTL8 did not have a large effect on vascular wall thickness relative to normal mice, and vascular wall thickness in mice in the CIH group was increased by about one-half of the normal thickness relative to the placebo group, which indicates that chronic intermittent hypoxia in sleep apnea pattern resulted in significant increase in vascular wall thickness in mice, whereas CIH + ANGPTL8 inhibited mice maintained vascular wall thickness in mice subjected to chronic intermittent hypoxia in sleep apnea pattern at normal levels due to the action of angiopoietin-like protein8 inhibitor. Therefore, inhibition of angiopoietin-like protein8 can significantly inhibit chronic intermittent hypoxia-induced vessel wall thickening in sleep apnea patterns.

The thoracic aorta sections of each group of mice were stained with collagen fiber and sirius red, respectively, to obtain fig. 4.

Wherein, the step of dyeing the collagen fiber comprises the following steps: dewaxing and washing the thoracic aorta section of each group of mice with water; dyeing for 40min by using potassium dichromate dyeing liquor; washing with water to remove loose color, and treating with acid fuchsin (2: 1) for 15 min; washing phosphomolybdic acid for 5min and 1% -acetic acid for 2-3 times; re-dyeing for 8min in brilliant green, washing for 2s with phosphomolybdic acid and 2-3 times with 1% acetic acid; dehydrated with absolute alcohol, xylene transparent, neutral gum-sealed, observed by upright microscope (Nikon, Tokyo, Japan) and image-collected, to obtain fig. 4-a to fig. 4-E.

As shown in fig. 4, fig. 4-a is a graph of collagen fiber staining of the blank control group, fig. 4-B is a graph of collagen fiber staining of the ANGPTL8 inhibition group, fig. 4-C is a graph of collagen fiber staining of the CIH group, and fig. 4-D is a graph of collagen fiber staining of the CIH + ANGPTL8 inhibition group, wherein the collagen fibers and reticular fibers are in a green dot or stripe shape; muscle fibers, cellulose, glial fibers, mucus, red blood cells, and the like are dotted or striped with bright pink colors of varying shades, and "400 x" in the above figures indicates a magnification of 400 x. FIG. 4-E is a schematic diagram showing a comparison of the collagen deposition levels in the four groups of mice, wherein the horizontal axis represents the groups, the vertical axis represents the collagen deposition level (%), the horizontal line at the top of the column represents the standard deviation of the collagen deposition levels in the mice, and the asterisks above the horizontal line indicate statistical significance.

As can be seen from fig. 4-a to 4-E, ANGPTL8 inhibited the level of collagen deposition in mice of the group relative to the blank control group, while the level of collagen deposition in mice of the CIH group was much higher than the blank control group, which indicates that chronic intermittent hypoxia in sleep apnea pattern can induce collagen deposition in the blood vessels of mice, but the level of collagen deposition in mice of the CIH + ANGPTL8 inhibited the level of collagen deposition in the blood vessels of mice of the blank control group, so that angiopoietin-like protein8 was inhibited in sleep apnea pattern.

The method for dyeing the sirius red comprises the following steps: removing the thoracic aorta section of each group of mice, placing in water, staining with picric acid for 15s, and changing the background into yellow; dripping and dyeing the slices for about 3min by adopting 1 part by mass of scarlet and 9 parts by mass of picric acid until the color of the slices is in a bright state; treating with anhydrous ethanol for 10 s; treating with xylene for 3 min; using clean gum, place on slides, do not seal, take 400 x photograph under polarizer, get fig. 4-F to fig. 4-J.

As shown in FIG. 4, FIG. 4-F is a photograph of sirius red staining of a blank control group, FIG. 4-G is a photograph of inhibition of sirius red staining of ANGPTL8, FIG. 4-H is a photograph of inhibition of sirius red staining of a CIH group, FIG. 4-I is a photograph of inhibition of sirius red staining of CIH + ANGPTL8, wherein "400X" in the above-mentioned photograph represents a magnification of 400 times, and FIG. 4-J is a comparative bar chart of collagen deposition levels of the above four groups of mice, wherein the horizontal axis represents the group, the vertical axis represents the collagen deposition activity level (%), the horizontal line at the top of the bar represents the standard deviation of collagen deposition levels of mice, and the asterisks above the horizontal line represent statistical significance.

As can be seen from fig. 4-F to fig. 4-J, ANGPTL8 showed no significant difference in the level of collagen deposition in mice in the group, compared to the placebo group, whereas mice in the CIH group showed a much higher level of collagen deposition than the placebo group, indicating that chronic intermittent hypoxia in sleep apnea mode induced collagen deposition in the blood vessels of mice, but that the level of collagen deposition in mice in the CIH + ANGPTL8 showed no significant difference in the level of collagen deposition in the control group, indicating that angiopoietin-like protein8 inhibited chronic intermittent hypoxia in sleep apnea mode induced collagen deposition in the blood vessels.

Respectively dyeing the thoracic aorta section of each group of mice with elastic fiber, namely blowing the section by electric blowing or baking the section until the section is dissolved with wax; dewaxing in xylene I for 15min, and sucking out the liquid with absorbent paper; dewaxing in xylene II for 15min, and sucking the liquid with absorbent paper; dewaxing in xylene III for 15min, and sucking the liquid with absorbent paper; washing with 100% alcohol for 5min, and drying with absorbent paper; washing with 95% ethanol for 5min, and drying with absorbent paper; washing with 80% ethanol for 5min, and drying with absorbent paper; washing with tap water for 2min, and washing off alcohol; adding iodine solution for 5min, and washing with tap water for 3 times; adding sodium thiosulfate for 5min, and washing with tap water for 3 times; adding 70% alcohol for 2 s; adding aldehyde fuchsin for 20s, and washing with 70% alcohol for 30 s; dyeing for 1s in orange G dye liquor, and cleaning for 3min by using absolute ethyl alcohol; adding xylene for 5 min; and (5) sealing the neutral gum. Images were observed and collected by upright microscope (Nikon, Tokyo, Japan) to obtain fig. 5.

As shown in fig. 5, fig. 5-a is a schematic view of staining spandex in a blank control group, fig. 5-B is a schematic view of staining spandex in an ANGPTL8 inhibition group, fig. 5-C is a schematic view of staining spandex in a CIH group, and fig. 5-D is a schematic view of staining spandex in a CIH + ANGPTL8 inhibition group, wherein "400 x" in the above-mentioned figures represents a magnification of 400 x, and fig. 5-E is a schematic view of comparing histograms of levels of degradation of elastin in sections of thoracic aorta of mice in the four groups, wherein the level of degradation of elastin in sections of thoracic aorta of mice in the blank control group is taken as 1, the horizontal axis represents a group, the vertical axis represents a level of degradation of elastin, the horizontal line at the top of the histograms represent a standard deviation of levels of degradation of elastin in mice, and the asterisks above the horizontal line represent statistical.

As can be seen from fig. 5, the level of elastin degradation in mice in the CIH group is much higher than that in the other three groups, which indicates that chronic intermittent hypoxia in sleep apnea can induce vascular wall elastic fiber disorders in mice compared to normal mice in the blank group, while the level of elastin degradation in mice in the CIH + ANGPTL 8-inhibited group is much lower than that in the CIH group, which is comparable to that in the blank group and ANGPTL 8-inhibited group, which indicates that angiopoietin-like protein8 can significantly inhibit chronic intermittent hypoxia-induced vascular wall elastic fiber disorders in sleep apnea.

Arterial blood pressure of each group of mice was measured by the tail-cover method using a non-invasive tail artery blood pressure measuring instrument and statistically analyzed using prism5.0(GraphPad Software, San Diego, CA), resulting in fig. 6.

As shown in fig. 6, fig. 6 is a schematic diagram of the variation of the systolic blood pressure (SBP, mmHg) of different age groups of mice in each group, wherein the horizontal axis represents the number of weeks of age of the mice, and the vertical axis represents the systolic blood pressure of the mice, it can be seen that after 6 weeks of age, namely after the mice in the CIH group start to be subjected to the chronic intermittent hypoxia treatment in the sleep apnea pattern, the systolic blood pressure of the mice in the CIH group is much higher than that of the mice in other three groups, so that the chronic intermittent hypoxia in the sleep apnea pattern can induce the blood pressure increase of the mice, while for the mice in the CIH + ANGPTL8 inhibition group, the angiopoietin-like protein8 neutralizing antibody is injected to inhibit the angiopoietin-like protein8, and the systolic blood pressure level of the mice in the sleep apnea pattern is not obviously different from that of the blank group, so that the angiopoietin-like protein8 can obviously reduce the blood pressure increase induced.

Respectively carrying out serum lipid detection on each group of mice, namely respectively taking a serum sample of each group of mice, adding 2.5 mu l of distilled water into a blank hole of a 96-well plate, and adding 2.5 mu l of a standard substance and 2.5 mu l of the serum sample into a calibration hole; adding 1180 mu l of R into each hole, mixing uniformly, incubating for 5min at 37 ℃, and determining each absorbance value A1 by using an enzyme-linked immunosorbent assay at 546nm wavelength; adding R260 μ l into each well, mixing, incubating at 37 deg.C for 5min, and measuring absorbance A2 with enzyme-labeling instrument at 546nm wavelength; colorimetry using an enzyme reader gave fig. 7 in which the lipid concentration (mmol/L) ═ was (sample a 2-sample a1) - (blank a 2-blank a 1)/(standard a 2-standard a1) - (blank a 2-blank a1) × the standard concentration (mmol/L).

As shown in fig. 7, fig. 7-a is a histogram comparing the serum low density lipoprotein cholesterol (LDL-C) concentrations of mice in the blank control group, ANGPTL8 inhibition group, CIH group, and CIH + ANGPTL8 inhibition group, and fig. 7-B is a histogram comparing the serum Total Cholesterol (TC) concentrations of mice in the blank control group, ANGPTL8 inhibition group, CIH group, and CIH + ANGPTL8 inhibition group, in which the horizontal axis represents the group, the vertical axis represents the concentrations of different lipids in the serum, the horizontal line at each column top represents the variance of the concentrations, and the uppermost horizontal line and asterisk in the figure represent the statistical significance of comparing the two groups covered by the horizontal line.

As can be seen from fig. 7, the levels of ldl cholesterol and total cholesterol in the serum of mice in the blank control group and the CIH group were significantly higher than normal values, while the levels of ldl cholesterol and total cholesterol in the serum of mice in the ANGPTL8 inhibition group and the CIH + ANGPTL8 inhibition group were significantly reduced, indicating that inhibition of angiopoietin-like protein8 can significantly reduce the chronic intermittent hypoxia-induced lipid elevation in the sleep apnea mode.

Each group of mice was subjected to immunohistochemical treatment and western blot detection treatment, respectively, to obtain fig. 8.

Taking out thoracic aorta vascular tissue sections of each group of mice, airing for 10min at room temperature, fixing for 30min with 4% paraformaldehyde, washing for 3 times with PBS (5 min each time), incubating for 20min with endogenous peroxidase inhibitor, washing for 3 times with PBS (5 min each time), sealing for 30min with serum, sucking dry serum, incubating with primary antibody (HIF-1 α, ab16066, 100 μ l, purchased from Abcam) at 4 deg.C overnight, airing for 30min at room temperature, incubating with secondary antibody (rabbit antibody or mouse antibody corresponding to the primary antibody) for 2 h, staining with DAB staining solution (positive brown), counterstaining with hematoxylin for about 30s-1min, washing with 1% hydrochloric acid alcohol, washing with tap water, returning blue, washing with water, placing the slices in a washing cabinet, sequentially placing the slices in 70% alcohol-80% alcohol-90% alcohol-95% alcohol-absolute ethyl alcohol I-absolute ethyl alcohol II-xylene I-II-xylene, dehydrating and placing each reagent in a 2min, ventilating, and observing the vertical stroke image with a microscope (8 kokon-8).

As shown in FIG. 8, FIG. 8-A is a photograph showing immunohistochemical staining of a blank control group of mice, FIG. 8-B is a photograph showing immunohistochemical staining of an ANGPTL 8-suppressed group of mice, FIG. 8-C is a photograph showing immunohistochemical staining of a CIH group of mice, and FIG. 8-D is a photograph showing immunohistochemical staining of a CIH + ANGPTL 8-suppressed group of mice, wherein "400X" in the above-mentioned photograph indicates a magnification of 400 times, FIG. 8-E is a bar graph showing the expression level of HIF-1 α in each group of mice, wherein the horizontal axis indicates the group, the vertical axis indicates the expression level of HIF-1 α, the horizontal line at the top of the bar indicates the standard deviation of the expression level of HIF-1 α, and the horizontal line at the top of the figure and the model above the horizontal line indicate that the two groups covered with the horizontal line are compared and have statistical significance.

As can be seen from FIGS. 8-A to 8-E, the expression level of HIF-1 α in mice in the CIH group was significantly higher than that in the other three groups, indicating that CIH significantly promoted the expression of HIF-1 α, while the expression of HIF-1 α in mice in the CIH + ANGPTL 8-inhibited group was not significantly different from that in the blank control group, indicating that inhibition of angiopoietin-like protein8 significantly reduced the increased expression of HIF-1 α induced by chronic intermittent hypoxia in the sleep apnea pattern.

The steps of the western blot detection process include: the thoracic aortic region of each group of mice was collected, frozen in liquid nitrogen, and stored at-80 ℃. The preparation of the total protein extraction experimental reagent and the tissue lysate are carried out in situ, the volume is calculated, the protease inhibitor (100X), the phosphatase inhibitor (100X) and the EDTA (100X) are added into the tissue protein extraction reagent, a mixer is used for mixing evenly, and the mixture is put on ice for standby, namely 10 mul of the protease inhibitor, 10 mul of the phosphatase inhibitor and 10 mul of the Ethylene Diamine Tetraacetic Acid (EDTA) are respectively added into 1ml of the lysate.

The blood vessel protein in the thoracic aorta region of each group of mice was extracted by homogenization, i.e., thoracic aorta tissue was taken, blood was washed with physiological saline, weighed, and placed on ice. Shearing the tissue, adding prepared lysate according to the ratio of 0.5ml of tissue lysate to 100mg of tissue, and grinding the tissue by using a homogenizer until no macroscopic fragments exist in the tissue; the milling process requires the operation of inserting a homogenizer on ice and minimizes the generation of air bubbles; sucking the tissue suspension into a 1.5ml EP tube, uniformly mixing for 15 seconds by using a mixer, placing on ice for 10 minutes, and repeating the steps for 3 times; centrifuging at 13000rpm for 15min at 4 deg.C, and collecting supernatant to a new centrifuge tube to complete total protein extraction.

The Protein concentration of each Protein sample was determined using the BCA Protein concentration Assay Kit (Pierce BCA Protein Assay Kit). The kit (Thermo Scientific, Prod 23228) comprises BCA reagent A, BCA reagent B and albumin standard reagent.

Equal amounts of protein were separated by 10% SDS-PAGE gel (lanes 40, etc.), blots were probed with anti-GAPDH (from Abcam; 1:1000 dilution), anti-ANGPTL 8 (from Abcam; 1:1000 dilution), anti-HIF-1 α (from Abcam; 1:1000 dilution) overnight at 4 deg.C, washed with Tris buffered saline containing Tween 20 and incubated with secondary antibody (ZSBG-) for 1 hour at room temperature for BIO, Beijing, China; 1: 10000), then blots were washed and analyzed using ChemiDoc (TM) TouchImaging System (Bio-Rad) to give FIGS. 8-F and 8-G.

As shown in FIG. 8, FIG. 8-F is a western-blot of relevant HIF-1 α levels in blood vessels of mice in the blank control group, ANGPTL8 inhibition group, CIH group and CIH + ANGPTL8 inhibition group, GAPDH is glyceraldehyde-3-phosphate dehydrogenase, i.e., an internal reference, black areas indicate the contents of HIF-1 α and glyceraldehyde-3-phosphate dehydrogenase in each group, the larger the black area is, the more HIF-1 α and glyceraldehyde-3-phosphate dehydrogenase are contained, FIG. 8-G is a bar graph of relevant HIF-1 α levels in blood vessels of mice in the blank control group, ANGPTL8 inhibition group, CIH group and CIH + ANGPTL8 inhibition group, the horizontal axis indicates the group, the vertical axis indicates the relevant-1 α level internally referenced by glyceraldehyde-3-phosphate dehydrogenase, the horizontal line at the top of the bar indicates the standard deviation of the relevant HIF-1 α level, and the horizontal line and asterisk in the graph indicate that the two groups are compared and the two groups have statistical significance.

As can be seen from FIGS. 8-F and 8-G, the relative HIF-1 α levels in mice in the CIH group were significantly higher than those in the other three groups, indicating that CIH significantly promoted HIF-1 α expression, while CIH + ANGPTL8 inhibited HIF-1 α expression in mice in the group was not significantly different from that in the placebo group, indicating that inhibition of angiopoietin-like protein8 significantly reduced the chronic intermittent hypoxia-induced increased HIF-1 α expression in sleep apnea.

Each group of mice was subjected to western blot detection treatment again, i.e., an equal amount of protein was separated by 10% SDS-PAGE gel (lanes such as 40 separation), blots were detected overnight at 4 ℃ using anti- β -actin (purchased from Abcam; 1:1000 dilution), anti-ANGPTL 8 (purchased from Abcam; 1:1000 dilution), anti-Bcl 2 (purchased from Cell signal; 1:1000 dilution), anti-Bax (purchased from Cell signal; 1:1000 dilution), washed with Tris buffered saline containing Tween 20, and incubated with a secondary antibody (ZSBG-) for 1 hour at room temperature, Beijing, China; 1: 10000), and blots were washed and analyzed using a ChemiDoc Touch imaging System (Bio-Rad), yielding FIG. 9.

FIG. 9A is a western-blot of the levels of Bcl2 and Bax in the blood vessels of mice in the blank control group, ANGPTL8 inhibition group, CIH group and CIH + ANGPTL8 inhibition group, wherein Bcl2 and Bax are both genes related to cell proliferation, β -actin is an internal reference, the black areas indicate the levels of Bcl2, Bax and β -actin in each group, the levels of Bcl2, Bax and β -actin are higher and the black areas are larger, FIG. 9B is a bar graph of the levels of Bcl2 and Bax in the blood vessels of the mice in the above four groups, wherein the horizontal axis indicates the group, the vertical axis indicates the level of the relevant Bcl2 and Bax, the horizontal line at the top of the bar indicates the standard deviation of the level of the relevant Bcl2 and Bax, and the asterisks above the horizontal line and the horizontal line at the top of the graph indicate that the two groups are compared to make statistical significance.

As can be seen from fig. 9, the Bcl2 level/Bax level of the CIH group mice is significantly higher than that of the other three groups, which indicates that chronic intermittent hypoxia in sleep apnea pattern can significantly promote the expression of Bcl2 and Bax, while CIH + ANGPTL8 inhibits the expression of HIF-1 α in the group mice to be not significantly different from that of the blank group, which indicates that inhibition of angiopoietin-like protein8 can significantly inhibit the chronic intermittent hypoxia-induced Bcl2 level and Bax level expression increase in sleep apnea pattern, i.e., inhibition of angiopoietin-like protein8 can significantly inhibit the chronic intermittent hypoxia-induced cell proliferation-related gene expression increase in sleep apnea pattern.

Each group of mice was subjected to Proliferating Cell Nuclear Antigen (PCNA) immunofluorescence staining and smooth muscle cell staining, respectively, to obtain fig. 10.

The step of immunofluorescence staining treatment comprises: the thoracic aorta sections of each group of mice were deparaffinized in water, 1% citric acid antigen repaired, 10% peroxidase treated for 10min, serum blocked for 20min, primary anti-PCNA (ab29, from Abcam, 1:200 dilution) incubated overnight, secondary airing for 30min, fluorescent secondary antibody (corresponding to primary anti-mouse antibody, A0460,1:200 dilution, 555 red, from beyond) incubated for 60min, DAPI mounting, Ni-UNikon Upper Microscope mounted with aDS-Ri2 color CCD (Nikon, Tokyo, Japan) sampling, and FIG. 10-A to FIG. 10-D were obtained.

As shown in FIG. 10, FIG. 10-A is a fluorescence staining pattern of proliferating cell nuclear antigen of a blank control group mouse, FIG. 10-B is a fluorescence staining pattern of proliferating cell nuclear antigen of an ANGPTL8 inhibiting group mouse, FIG. 10-C is a fluorescence staining pattern of proliferating cell nuclear antigen of a CIH group mouse, and FIG. 10-D is a fluorescence staining pattern of proliferating cell nuclear antigen of a CIH + ANGPTL8 inhibiting group mouse, in which a blue dotted block portion is proliferating cell nuclear antigen, and "400X" in the above-mentioned figures represents a magnification of 400 times.

As can be seen from fig. 10-a to fig. 10-D, the proliferating cell nuclear antigen in the CIH group was significantly increased compared to the other three groups, indicating that chronic intermittent hypoxia in sleep apnea pattern can significantly promote the expression of proliferating cell nuclear antigen in the vascular wall, while the CIH + ANGPTL8 inhibition group proliferating cell nuclear antigen was not significantly different from the blank control group proliferating cell nuclear antigen, indicating that inhibition of angiopoietin-like protein8 can significantly inhibit the chronic intermittent hypoxia-induced increase in proliferating cell nuclear antigen expression in sleep apnea pattern.

The step of staining the smooth muscle cells included deparaffinizing the thoracic aorta sections of each group of mice in water, 1% citric acid antigen retrieval, 10% peroxidase 10min, serum blocking for 20min, incubation overnight with primary antibody α -SMA (ZM0003, available from China fir bridge, 1:200 dilution), next day air slide for 30min, secondary antibody (corresponding to the primary antibody, PV-9000, available from China fir bridge, 1:200 dilution) for 60min, DAB (DAB-0031, available from Meristine) color development, neutral gum blocking, Ni-UNikon Uperlight microscope applied with a DS-Ri2 color CCD (Nikon, Tokyo, Japan) sampling, yielding FIGS. 10-E to 10-H.

As shown in FIG. 10, FIG. 10-E is a graph showing staining of mouse smooth muscle cells in a blank control group, FIG. 10-F is a graph showing staining of mouse smooth muscle cells in an ANGPTL8 inhibition group, FIG. 10-G is a graph showing staining of mouse smooth muscle cells in a CIH group, FIG. 10-H is a graph showing staining of mouse smooth muscle cells in a CIH + ANGPTL8 inhibition group, wherein "400X" in the above graphs indicates a magnification of 400 times, and FIG. 10-I is a histogram comparison of expression levels of the four groups of mouse vascular smooth muscle cells, wherein the horizontal axis indicates the group, the vertical axis indicates the expression level (%) of the vascular smooth muscle cells, the horizontal line at the top of the histogram indicates the standard deviation of the expression level of the vascular smooth muscle cells, and the model above the horizontal line indicates statistical significance.

As can be seen from fig. 10-E to fig. 10-I, the expression of vascular smooth muscle cells in the CIH group is significantly increased compared to the other three groups, which indicates that chronic intermittent hypoxia in sleep apnea pattern can significantly promote the expression of vascular smooth muscle cells in the vascular wall, while CIH + ANGPTL8 inhibits the vascular smooth muscle cells in group from being significantly different from the vascular smooth muscle cells in the blank control group, which indicates that inhibition of angiopoietin-like protein8 can significantly inhibit the increase of vascular smooth muscle cell expression induced by chronic intermittent hypoxia in sleep apnea pattern, i.e., inhibition of angiopoietin-like protein8 can significantly inhibit the proliferation of vascular smooth muscle cells induced by chronic intermittent hypoxia in sleep apnea pattern.

In summary, the application of the substance for inhibiting angiopoietin-like protein8 provided by the present application, wherein the substance for inhibiting angiopoietin-like protein8 can effectively prevent or alleviate pathological changes of the vascular wall induced by sleep apnea by inhibiting the thickening of the vascular wall, collagen deposition in the vascular wall cells, vascular wall elastic fiber disorder, blood pressure increase, blood lipid increase, expression of human hypoxia-inducible factor, expression of cell proliferation-related genes, expression of proliferating cell nuclear antigen, and expression of vascular smooth muscle cells in sleep apnea mode, so as to prevent or treat diseases related to sleep apnea-induced vascular remodeling, therefore, the substance for inhibiting angiopoietin-like protein8 can be applied to the products for preventing or treating sleep apnea-induced vascular remodeling, so as to effectively improve the therapeutic effect of sleep apnea-induced vascular remodeling and related diseases, the application prospect is good.

In this document, "upper", "lower", "front", "rear", "left", "right", and the like are used only to indicate relative positional relationships between relevant portions, and do not limit absolute positions of the relevant portions.

In this document, "first", "second", and the like are used only for distinguishing one from another, and do not indicate the degree and order of importance, the premise that each other exists, and the like.

In this context, "equal", "same", etc. are not strictly mathematical and/or geometric limitations, but also include tolerances as would be understood by a person skilled in the art and allowed for manufacturing or use, etc.

Unless otherwise indicated, numerical ranges herein include not only the entire range within its two endpoints, but also several sub-ranges subsumed therein.

The preferred embodiments and examples of the present application have been described in detail with reference to the accompanying drawings, but the present application is not limited to the embodiments and examples described above, and various changes can be made within the knowledge of those skilled in the art without departing from the concept of the present application.

Claims

1. Application of substance inhibiting angiopoietin-like protein8 in preparing product for preventing or treating diseases related to vascular remodeling induced by sleep apnea is provided.

2. The use of claim 1, wherein the product for preventing or treating a sleep apnea-induced vascular remodeling-related disease comprises a product for preventing or treating a sleep apnea pattern of chronic intermittent hypoxia-induced vascular remodeling-related disease.

3. The use of claim 1 or 2, wherein the prevention or treatment of a sleep apnea-induced vascular remodeling-related disease comprises inhibition of sleep apnea pattern chronic intermittent hypoxia-induced vessel wall thickening, collagen deposition within vessel wall cells, vessel wall elastic fiber disorders, elevated blood pressure, elevated blood lipids, expression of human hypoxia-inducible factors, expression of cell proliferation-related genes, expression of proliferating cell nuclear antigens, proliferation of vascular smooth muscle cells, and any combination thereof.

4. Use according to any of claims 1 to 3, wherein the substance inhibiting angiopoietin-like protein8 is used in combination with other drugs preventing or treating sleep apnea-induced vascular remodeling in said product.

5. Use according to any one of claims 1 to 4 wherein the substance that inhibits angiopoietin-like protein8 comprises an angiopoietin-like protein8 inhibitor.

6. Use according to claim 5, wherein the inhibitor of angiopoietin-like protein8 comprises an agent that binds to or interacts with angiopoietin-like protein8 in vivo or in vitro to inhibit the biological function of angiopoietin-like protein 8.

7. The use according to claim 6, wherein the inhibitor of angiopoietin-like protein8 comprises any one or a combination of angiopoietin-like protein8 antibodies, small molecule angiopoietin-like protein8 antagonists, nucleic acid-based inhibitors of angiopoietin-like protein8 expression or activity, peptide-based molecules that specifically interact with angiopoietin-like protein8, receptor molecules that specifically interact with angiopoietin-like protein8, proteins comprising ligand binding portions of low density lipoprotein receptors, scaffold molecules that bind angiopoietin-like protein8, fibronectin based scaffold constructs, other scaffold molecules based on naturally occurring repeat sequence proteins, and anti-angiopoietin-like protein8 aptamers.

8. Application of substance for knocking down or knocking out angiopoietin-like protein8 expression gene in preparing product for inhibiting vascular remodeling induced by sleep apnea.