CN110694067A

CN110694067A - Application of substance for inhibiting angiopoietin-like protein8

Info

Publication number: CN110694067A
Application number: CN201911076881.4A
Authority: CN
Inventors: 秦彦文; 焦晓璐
Original assignee: Beijing Anzhen Hospital
Current assignee: Beijing Anzhen Hospital
Priority date: 2019-11-06
Filing date: 2019-11-06
Publication date: 2020-01-17

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 hypertension and/or diseases related to vascular remodeling is provided. The application of the substance for inhibiting the angiopoietin-like protein8, which is provided by the application, can inhibit the endoplasmic reticulum stress pathway of smooth muscle cells and inhibit the proliferation and migration of the smooth muscle cells by inhibiting the collagen deposition of the substance for inhibiting the angiopoietin-like protein8, so that the blood pressure and the thickness of the vascular wall can be effectively reduced, namely, the hypertension and the vascular remodeling can be effectively prevented and treated, therefore, the substance for inhibiting the angiopoietin-like protein8 is applied to the product for preventing or treating the hypertension and/or the vascular remodeling, the treatment effect of the hypertension and the 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

Hypertension (hypertension) is a clinical syndrome characterized by an increase in systemic arterial blood pressure (systolic pressure and/or diastolic pressure) (systolic pressure not less than 140 mm hg, diastolic pressure not less than 90mm hg), which may be accompanied by functional or organic damage to organs such as heart, brain, kidney, etc.

The relaxation state of blood vessels, the blood flow distribution in the lumen and the flow speed of blood in the lumen can change at any time along with the metabolism and the needs of the body. With respect to such significant changes in hemodynamics, the structure of the vessel wall is relatively stable within a certain range; while as the blood vessels grow and the aging process progresses, some significant changes in their structure occur. This change is often a longer-term response of the vessel wall to alterations in hemodynamics and humoral and local endocrine factors, and this change in the conformation and function of the vessel under certain conditions is called vascular remodeling.

Vascular remodeling in hypertension (vascular remodelling) refers to the adaptive alteration of arterial vessel structure and function in a long-term hypertensive setting. Recent studies have found that hypertensive vascular remodeling is mainly manifested by hypertrophy and migration of medial smooth muscle cells, apoptosis and transformation of adventitial fibroblasts, inflammatory responses, extracellular fibrous matrix deposition, and the like. The vascular remodeling of hypertension is the change of vascular structure caused by hypertension, is a remarkable pathological feature, and is an important pathological basis for the development and deterioration of hypertension and the damage of target organs.

At present, various medicines for lowering blood pressure such as angiotensin II inhibitors, angiotensin II receptor antagonists, calcium channel inhibitors, diuretics, and the like are clinically available; the control of hypertension is still poor and the incidence of hypertension and its resulting vascular remodeling increases year by year. Therefore, the target for early prevention or intervention of the process is searched, the vascular remodeling of hypertension is reversed, and the method has very important significance for reducing the damage of target organs, improving the prognosis of patients, reducing the fatality rate of patients and reducing the national medical cost.

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 provides an application of a substance inhibiting angiopoietin-like protein8 in preparing a product for preventing or treating hypertension and/or diseases related to vascular remodeling.

Further wherein the product for preventing or treating vascular remodeling is a product for preventing or treating a hypertensive vascular remodeling-related disease.

Further, wherein the substance that inhibits angiopoietin-like protein8 prevents or treats hypertension and/or diseases associated with hypertensive vascular remodeling by inhibiting collagen deposition, inhibiting endoplasmic reticulum stress, inhibiting vascular smooth muscle cell proliferation and migration.

Further wherein said substance inhibiting angiopoietin-like protein8 is used in combination with other agents for the prevention or treatment of hypertension and/or vascular remodeling in said 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 provides application of the substance inhibiting the angiopoietin-like protein8 in preparing a product for inhibiting hypertension and/or vascular remodeling.

The application also provides application of the substance inhibiting the expression of the angiopoietin-like protein8 in preparing a product inhibiting hypertension and/or vascular remodeling.

The application also provides application of the substance for knocking down or knocking out the expression gene of the angiopoietin-like protein8 in preparing products formed by hypertension and/or vascular remodeling.

The application of the substance for inhibiting the angiopoietin-like protein8, provided by the application, can be used for inhibiting the angiopoietin-like protein8, inhibiting the endoplasmic reticulum stress pathway of smooth muscle cells and inhibiting the proliferation and migration of the smooth muscle cells by slowing down the collagen deposition in the smooth muscle cells, so that the blood pressure and the thickness of the vascular wall can be effectively reduced, namely the hypertension and the vascular remodeling can be effectively prevented and treated, therefore, the substance for inhibiting the angiopoietin-like protein8 is applied to the product for preventing or treating the hypertension and/or the vascular remodeling, the treatment effect of the hypertension and the vascular remodeling can be effectively improved, and the application prospect is good.

Drawings

FIG. 1 is a graph comparing the levels of angiopoietin-like protein8 in human serum as described in one embodiment of the present application;

FIG. 2 is a graph comparing the expression levels of angiopoietin-like protein8 in mouse vascular tissue as described in an embodiment of the present application;

FIG. 3 is a graph comparing the expression levels of angiopoietin-like protein 8mRNA in mouse vascular tissue as described in one embodiment of the present application;

FIG. 4 is a graph comparing immunohistochemical staining of mouse vascular tissue according to one embodiment of the present application;

FIG. 5 is a graph of co-localized staining of mouse angiopoietin-like protein8 with smooth muscle cells according to an embodiment of the present application;

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

FIG. 7 is a graph comparing HE staining of a vascular wall of a mouse according to an embodiment of the present application;

FIG. 8 is a graph comparing the staining of collagen fibers in mouse vascular tissue according to an embodiment of the present application;

FIG. 9 is a graph comparing the levels of mRNA associated with endoplasmic reticulum stress factor in mouse vascular tissue as described in one embodiment of the present application;

FIG. 10 is a graph comparing the levels of human vascular smooth muscle cell angiopoietin-like protein8 according to an embodiment of the present application;

FIG. 11 is a graph comparing the antigen content of proliferating cell nuclei of human vascular 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.

Angiotensin II (Angiotensin II, AngII): is a polypeptide substance produced by the hydrolysis of angiotensin I under the action of angiotensin converting enzyme. Angiotensin receptors are present on the vascular smooth muscle, adrenal cortico zonal cells, and cells of several parts of the brain, heart and kidney organs of the human body. Angiotensin II binds to angiotensin receptor to cause corresponding physiological effects, including constriction of systemic arteriole and vein, increase of blood pressure, and increase of blood volume in heart; increasing the amount of sympathetic vasoconstrictor neurotransmitter release; tone the sympathetic vasomotor center; stimulation of the adrenal gland to synthesize and release aldosterone and to cause or enhance craving, resulting in drinking behavior. Angiotensin II is commonly measured in plasma by radioimmunoassay.

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.

The Western Blot technique comprises the following steps:

1. cleaning glass sheets

2. Glue preparation

Separating glue: 10% of the separation gel is suitable for molecules with large molecular weight, and 12% of the separation gel is suitable for molecules with small molecular weight.

Concentrating the glue:

preparing separation gel, adding other components in the preparation process of the separation gel, adding TEMWD, mixing well, rapidly adding into glass plate, and spraying liquid surface with alcohol to flatten the liquid surface (30 min).

Preparing concentrated gel, pouring out alcohol after the gel is separated, adding the concentrated gel into a glass plate, immediately inserting the comb teeth prepared in advance, and standing for 1 h.

3. Electrophoresis

Two plates are placed in an electrophoresis tank, and if only one gel is run by adding prepared 1X electrophoresis buffer solution between the two plates, the other side is inserted into the electrophoresis tank by using a transparent gel plate (a dummy plate).

1) Flat pull-out comb (two hands respectively hold the comb and pull it out vertically and lightly)

2) Sample application

i. Adding a protein maker: 3/5/7/10ul (depending on the loading condition, the method of distinguishing the left and right can be selected flexibly, and different quantities of marker can be added to the left and right to distinguish the left and right).

ii. Determining the amount of sample loading

a. Loading the sample with stock solution: the volume of the upper 30ug (or 15ug/20ug/25ug) protein sample was calculated from the concentration for loading.

b. Diluting the stock solution: different sample proteins are diluted into a system with the same concentration, and the same volume is loaded.

3) And (5) covering the cover, wherein black is opposite to black, and red is opposite to red.

4) Firstly, keeping the pressure constant at 80V/90V to make the sample run in the concentrated gel; when the separation gel is discharged, the voltage is changed to 100V/120V.

5) And when the blue protein liquid runs to the bottom of the gel, ending electrophoresis and turning off the power supply.

4. Rotary film

1) 1X electrotransfer solution (1000ml) is prepared, and then the mixture is rapidly cooled at-80 ℃ or is prepared in advance and then is cooled at 4 ℃.

Wherein, D2H 2O: methanol: 10X electrotransfer solution ═ 7:2:1(700ml, 200ml, 100ml)

2) And respectively soaking the film transferring clamp and the film in the electrotransformation liquid.

3) The glass plate is pried to remove the stripping glue, after the small glass plate is removed, the upper part, the lower part and the left part and the right part of the glue are slightly cut off, the separation glue is prevented from being scraped, and the separation glue is carefully stripped to cover the membrane.

4) Rolling off bubbles below the filter paper on the whiteboard side of the clamp, transferring the film and the glue onto the filter paper on the whiteboard side (white film black glue), adjusting to align the film and the filter paper, and slightly rolling off the bubbles; covering 3 pieces of filter paper on the glue and removing bubbles; finally, another spongy cushion is covered, and the clamp is closed.

5) Putting ice in the basin, putting the clamp into the electric rotary tank, wherein the end of the clamp is upward, the black surface faces the black surface of the tank, and the white surface faces the red surface of the tank; pouring the electric conversion solution; setting: 300mA, 120 min.

5. Sealing of

1) And (3) immediately putting the membrane into 1X TBST for rinsing for 1-2min after the membrane transferring is finished, and washing the membrane transferring liquid on the membrane.

2) The membrane was placed in milk (5% skimmed milk powder 2.5g +1X TBST to 50ml and shaken on a shaker for 60min to homogenize completely.

3) Washing the membrane 3 times and 5-10 min/time with 1X TBST.

6. Primary antibody incubation:

i. diluting the primary antibody: a total of 3ml was diluted with primary anti-diluent.

ii, film shearing: according to the principle of non-cross incubation and anti-tumor, the membranes of the bands of different target proteins and internal references are cut, the cut bands are placed in boxes with grids, the anti-tumor or the internal references are incubated respectively, and the shaking table is used for overnight at 4 ℃.

7. Incubation secondary antibody

i. The next day, the membrane was removed, 1X TBST (submerged membrane) was added, the membrane was washed 5-10min X4 times on a shaker, and the primary antibody was recovered at-20 ℃.

Dilute secondary antibody with 1X TBST (1: 10000).

And iii, oscillating the secondary antibody in a shaking table at room temperature for incubation for 1.5-2h to ensure that the secondary antibody is completely uniform.

Wash 10min X4 times in 1X TBST.

8. Developed using a ChemiDocTMTouch imaging System (Bio-Rad).

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.

OCT embedding agent (optimal cutting temperature compound): is a water-soluble mixture of polyethylene glycol and polyvinyl alcohol, which is widely used in immunohistochemical laboratories to support tissue during cryosectioning to increase tissue continuity and reduce wrinkling and fragmentation.

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.

Transcriptome sequencing technology (RNA-seq): mRNA, smallRNA, NONcoding RNA and the like or some of the mRNA, the smallRNA, the NONcoding RNA and the like are sequenced by using a high-throughput sequencing technology to reflect the expression level of the mRNA, the smallRNA, the NONcoding RNA and the like.

Quantitative PCR: also known as Quantitative real time polymerase chain reaction (Q-PCR/qPCR/rt-qPCR, Quantitative real-time PCR, real-time Quantitative PCR), is a method and technology for detecting the total amount of products after each Polymerase Chain Reaction (PCR) cycle by using a fluorescent dye in a DNA amplification reaction, and has broad and narrow concepts. The quantitative PCR technique in a broad concept refers to the quantification of the amount of a PCR starting template by analyzing a PCR end product or monitoring a PCR process using an external reference or an internal reference as a standard. The quantitative PCR technology (strictly quantitative PCR technology) in the narrow sense means that the aim of accurately quantifying the number of the initial templates is achieved by monitoring the PCR process (monitoring the amplification efficiency) by using an external standard method (the specificity is ensured by a fluorescent hybridization probe), and meanwhile, false negative results are effectively eliminated by using an internal control (the amplification efficiency is zero).

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.

Endoplasmic Reticulum Stress (ERS): it is a state of homeostasis imbalance in the endoplasmic reticulum caused by various factors and is considered as one of the initial reactions of cells in a stress state. Mainly including Unfolded Protein Response (UPR), endoplasmic reticulum overload response and steroid regulation cascade 3. Three ERS-sensing UPR signaling pathways have been discovered, the myokinase 1a (IRE1a) signaling pathway, the double-stranded RNA-dependent protein kinase-like endoplasmic reticulum kinase (PERK) signaling pathway, and the activated transcription factor 6(ATF6) signaling pathway.

The embodiment provides application of a substance inhibiting angiopoietin-like protein8 in preparing a product for preventing or treating hypertension and/or diseases related to vascular remodeling.

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 hypertension and/or vascular remodeling related diseases comprises one or any combination of more of a product for preventing hypertension, a product for preventing hypertension related diseases, a product for preventing vascular remodeling related diseases, a product for treating hypertension related diseases, a product for treating vascular remodeling and a product for treating vascular remodeling related diseases. The related diseases are diseases generated in the process of forming hypertension and/or vascular remodeling, or diseases such as complications and sequelae caused by hypertension and/or vascular remodeling and have a certain correlation with hypertension and/or 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.

Specifically, the product for preventing or treating the hypertensive vascular remodeling-related disease is a medicament, a preparation, a kit and/or an apparatus and the like which have a certain treatment effect on the hypertensive vascular remodeling-related disease.

Specifically, the other drugs for preventing or treating hypertension and/or vascular remodeling may be diuretic hypotensive drugs, sympathetic suppressive drugs, renin-angiotensin system suppressive drugs, calcium antagonists, vasodilators, etc., which are not limited in the present application.

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 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, trigonal tetrapeptide repeat protein (tetratricopeptide repeat proteins), etc.

The present example also provides the use of an agent that inhibits angiopoietin-like protein8 in the preparation of a product that inhibits hypertension and/or vascular remodeling.

Specifically, the product for inhibiting the formation of hypertension and/or vascular remodeling is a drug, a preparation, a kit and/or an apparatus and the like for inhibiting the occurrence or development of hypertension and/or vascular remodeling.

The present invention also provides the use of a substance that inhibits angiopoietin-like protein8 expression in the preparation of a product for inhibiting hypertension and/or 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 expression gene of the angiopoietin-like protein8 in preparing products formed by hypertension and/or 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 embodiments also provide a method for preventing and/or treating hypertension and/or a vascular remodeling-related disease, comprising preventing and/or treating hypertension and/or a vascular remodeling-related disease by inhibiting angiopoietin-like protein 8.

This example is further illustrated below with reference to specific experiments.

Expression of angiopoietin-like protein8 in human hypertensive patients

Patients with primary hypertension (according to the diagnosis standard of Chinese hypertension prevention and treatment guidelines that SBP is not less than 140 and/or DBP is not less than 90mmHg in a diagnosis room, and hypertension can be diagnosed by measuring the blood pressure of three times of non-same day according to the standard) are continuously collected and diagnosed in a hypertensive department of a certain hospital, patients with secondary hypertension such as kidney diseases, pheochromocytoma, primary aldosteronism and the like, patients with severe arrhythmia, patients with moderate and severe cardiac insufficiency, active liver dysfunction are excluded, or patients with glutamic-pyruvic transaminase (ALT) or glutamic-oxalacetic transaminase (AST) more than 3 times of the upper limit of normal, patients with malignant diseases such as tumor, anemia, acute and chronic inflammatory diseases such as rheumatic arthritis and rheumatoid arthritis, and the rest 80 patients with essential hypertension are used as an observation group.

Randomly selecting 80 subjects from the population with normal blood pressure and health examination in the same time period of the same hospital examination center as a normal control group, and recording the conditions of age, ethnicity, sex, weight, native place, smoking, height, drinking and the like in detail by adopting a questionnaire survey method; all patients were rested for 5 minutes and blood pressure was measured. Measuring the blood pressure of the right upper arm for 3 times, and calculating an average value; collecting the detection results of multiple biochemical indexes such as blood fat, blood sugar, liver and kidney functions, blood routine and the like, collecting the auxiliary examination results such as carotid artery ultrasound, echocardiography and the like, and carefully inquiring the disease history such as cardiovascular diseases, diabetes, liver and kidney diseases, family history and the like.

After 8-10 hours fasting, 10ml blood samples were taken intravenously from 80 patients in the observation group and 80 subjects in the normal control group, and after centrifuging the blood samples at 3000rpm for 10 minutes, serum was extracted and frozen in a refrigerator at-80 ℃.

The amount of angiopoietin-like protein8 in the serum of the patients in the observation group and the subjects in the normal control group was determined by a high-sensitivity sandwich immunoassay (ELISA) (kit from EK-051-60; PhoenixPharmaceuticals, Inc., Burlingame, Calif.). The angiopoietin-like protein8 antibody in the high-sensitivity sandwich immunoassay kit is coated in a 96-hole microplate to prepare a solid phase carrier. Adding a standard substance or a specimen into each micropore, wherein the angiopoietin-like protein8 is combined with an antibody connected to a solid phase carrier, then adding a biotinylated angiopoietin-like protein8 antibody, washing the unbound biotinylated antibody, adding horseradish peroxidase (HRP) -labeled avidin, washing thoroughly again, and adding a 3,3',5,5' -Tetramethylbenzidine (TMB) substrate for color development. TMB is converted to blue by the catalysis of peroxidase and to the final yellow by the action of an acid. The shade of the color was positively correlated with angiopoietin-like protein8 in the sample. The absorbance (o.d. value) was measured at 450nm wavelength with a microplate reader to obtain a standard curve, and then the concentration of angiopoietin-like protein8 was calculated from the standard curve and statistically analyzed using SPSS software (IBM corp., Armonk, NY, USA) to obtain fig. 1.

FIG. 1 is a bar graph comparing the content of angiopoietin-like protein8 in the serum of the patients in the observation group and the subjects in the normal control group, wherein the horizontal axis represents the group, the vertical axis represents the content (ng/ml) of angiopoietin-like protein8, the horizontal line above the bar represents the standard deviation of the content of angiopoietin-like protein8, and the asterisk indicates that the experiment and the experimental results have statistical significance. It can be seen that the content of angiopoietin-like protein8 in the serum of the patients in the observation group is obviously greater than that of angiopoietin-like protein8 in the serum of the subjects in the normal control group, so that the expression level of angiopoietin-like protein8 in the serum of the patients with primary hypertension is obviously increased, and the angiopoietin-like protein8 is activated in the process of vascular remodeling of human hypertension.

Second, the expression and localization of angiopoietin-like protein8 in mouse vascular tissue

40C 57BL/6 mice (purchased from Beijing Wittingle laboratory animals Co., Ltd.) were selected for 8-10 weeks, divided into two groups, namely a normal saline infusion group and an angiotensin II infusion group, wherein each group comprises 20 mice, and the mice are fed with common feed.

The mice of the normal saline perfused group and the angiotensin II perfused group are respectively injected with the normal saline and the angiotensin II by adopting a subcutaneous micro-pump injection method, wherein the dose of the angiotensin II injected by each mouse of the angiotensin II perfused group is 490ng/kg/min by adopting a micro-pump, the injection lasts for 14 days, and the angiotensin II is purchased from Sigma company.

Carrying out anesthesia treatment on mice of each group by injecting 1% pentobarbital (60mg/kg) into abdominal cavities, after anesthesia, disinfecting skin and opening abdomen, quickly opening the chest cavity of the mice, taking blood from the apex of the heart, quickly taking out tissues of the liver, the heart and the kidney after perfusion of normal saline, and quickly freezing in liquid nitrogen; fully opening the pleuroperitoneal cavity of the mouse, stripping off redundant tissues, fully exposing aorta blood vessels, fully separating tissues around the blood vessels under a body microscope, putting the blood vessels into liquid nitrogen for quick freezing, and then putting the tissues into a refrigerator at the temperature of 80 ℃ below zero for later use.

The method comprises the following steps of respectively detecting the protein levels of angiopoietin-like protein8 in heart, kidney, liver and vascular tissues of mice in a normal saline perfusion group and an angiotensin II perfusion group by adopting a Western Blot technology, wherein the detection step of the Western Blot technology comprises the following steps: cleaning the glass plate; preparing glue; electrophoresis; film transferring; sealing; primary antibody incubation, wherein the primary antibody is angiopoietin-like protein8 (purchased from Abcam, usa); incubation secondary antibody (purchased from Abcam, usa); for visualization, details can be found in the "Western immunoblot" noun interpretation section above and imaged using the ChemiDocTMTouch imaging System (Bio-Rad) to obtain FIG. 2.

As shown in FIG. 2, FIG. 2-A is a western-blot of angiopoietin-like protein8 in liver tissue of mice in the saline and angiotensin II perfused groups, FIG. 2-B is a western-blot of angiopoietin-like protein8 in kidney tissue of mice in the saline and angiotensin II perfused groups, FIG. 2-C is a western-blot of angiopoietin-like protein8 in heart tissue of mice in the saline and angiotensin II perfused groups, FIG. 2-D is a western-blot of angiopoietin-like protein8 in blood vessel tissue of mice in the saline and angiotensin II perfused groups, wherein GAPDH is glyceraldehyde-3-phosphate dehydrogenase (glyceraldehyde-3-phosphate dehydrogenase), i.e., an internal reference, and black areas indicate the contents of angiopoietin-like protein8 and glyceraldehyde-3-phosphate dehydrogenase in each group, the larger the black area, the more angiopoietin-like protein8 and glyceraldehyde-3-phosphate dehydrogenase are contained in the liver, kidney, heart or vascular tissue of the mouse, and dalton (KD) is atomic mass unit. Fig. 2-E are bar graphs comparing the protein levels of liver, kidney, heart and vascular tissue angiopoietin-like protein8 of mice in the saline-infused group and angiotensin II-infused group, wherein the horizontal axis represents the group, the vertical axis represents the protein level of angiopoietin-like protein8 (with glyceraldehyde-3-phosphate dehydrogenase as a reference), the horizontal line at the top of the bar graph represents the standard deviation of the protein level of angiopoietin-like protein8, and the top horizontal line and asterisk in the figure represent the comparison between the two groups covered by the horizontal line, which is statistically significant.

As can be seen from fig. 2, there was no significant difference in the protein levels of angiopoietin-like protein8 in liver, kidney and heart between the mice in the angiotensin II perfused group and the mice in the normal saline perfused group, but the protein level of angiopoietin-like protein8 in the vascular tissue of the mice in the angiotensin II perfused group was significantly higher than that of angiopoietin-like protein8 in the vascular tissue of the mice in the normal saline perfused group.

The expression levels of angiopoietin-like protein 8mRNA in liver, kidney, heart and vascular tissues of the saline perfusate and angiotensin II perfusate were measured by RT-PCR, and statistically analyzed by prism5.0(graphpad software, San Diego, CA), to obtain fig. 3.

As shown in fig. 3, fig. 3-a is a histogram comparing the mRNA levels of angiopoietin-like protein8 in the liver, kidney, heart and vascular tissues of mice in the saline-perfused group and angiotensin II-perfused group, wherein the mRNA level of angiopoietin-like protein8 in the liver of mice in the saline-perfused group is 1, the mRNA levels of angiopoietin-like protein8 in the liver, kidney, heart and vascular tissues of other groups of mice are based on the mRNA level of angiopoietin-like protein8 in the liver of mice in the saline-perfused group on average, the horizontal axis represents the group classification, the vertical axis represents the mRNA level of angiopoietin-like protein8, the horizontal line at the top of the bar represents the standard deviation of the mRNA level of angiopoietin-like protein8, and the uppermost horizontal line and asterisk in the graph represent that the two groups covered by the horizontal line are compared, and have statistical significance. Fig. 3-B is a histogram comparing the mRNA levels of angiopoietin-like protein8 in vascular tissues of mice in saline-perfused group and angiotensin II-perfused group, in which the mRNA level of angiopoietin-like protein8 in vascular tissues of mice in saline-perfused group is 1, the mRNA level of angiopoietin-like protein8 in vascular tissues of mice in angiotensin II-perfused group is referred to the saline-perfused group, the horizontal axis represents the group, the vertical axis represents the mRNA level of angiopoietin-like protein8, the horizontal line at the top of the histogram represents the standard deviation of the mRNA level of angiopoietin-like protein8, and the uppermost horizontal line and the asterisk in the graph represent the comparison between the two groups covered by the horizontal line, and are statistically significant.

As can be seen from FIG. 3, the mRNA of angiopoietin-like protein8 in liver, kidney and heart of mice in angiotensin II perfused group was not significantly different from that of mice in normal saline perfused group, but the level of mRNA of angiopoietin-like protein8 in vascular tissue of mice in angiotensin II perfused group was significantly higher than that of angiopoietin-like protein8 in vascular tissue of mice in normal saline perfused group.

The protein level and mRNA level of angiopoietin-like protein8 in the vascular tissue of mice in the angiotensin II perfused group are obviously higher than those of mice in the normal saline perfused group, so that the expression of angiopoietin-like protein8 in the vascular tissue of mice in the angiotensin II perfused group is obviously increased, and the angiopoietin-like protein8 in the vascular tissue of mice suffering from hypertension can be inferred to be obviously increased because the angiotensin II micropump is perfused into a classical mouse model recognized by hypertension.

After the mouse is anesthetized, the blood vessel of the mouse is perfused by heparin saline, and residual blood in the blood vessel of the mouse is removed. The mouse heart and the proximal aortic tissue were placed in 4% paraformaldehyde overnight at 4 ℃, then transferred to a 30% sucrose solution overnight at 4 ℃, the vascular tissue was taken out of the sucrose solution, blotted, embedded with OCT, and cryosectioned. A plurality of continuous 7-micron-thick frozen sections of each group of mice from the middle of the ventricle to the aortic arch were collected and stored at-20 ℃.

The angiopoietin-like protein8 in the vascular tissue sections of mice in the angiotensin II perfusion group and the normal saline injection group are respectively subjected to immunohistochemical staining, namely the vascular tissue sections of each group of mice are taken out, dried at room temperature for 10min, fixed by 4% paraformaldehyde for 30min, washed by PBS for 3 times, 5min each time, incubated by an endogenous peroxidase inhibitor for 20min, washed by PBS for 3 times, 5min each time, sealed by serum for 30min, and subjected to serum suction drying, and then primary antibody (ANGPTL8, purchased from Abcam) is kept overnight at 4 ℃. The next day, after drying in the air for 30min at room temperature, after incubating the corresponding secondary antibody for one hour, staining with DAB color development solution (brown is positive), re-staining with hematoxylin for about 30s-1min, washing with water, differentiating with 1% hydrochloric acid, and washing with tap water to turn blue. After the slices were washed 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 dried in a fume hood, and then the neutral gum was mounted, and observed and images were collected with an upright microscope (Nikon, Tokyo, Japan), to obtain fig. 4.

As shown in fig. 4, fig. 4-a is a graph showing immunohistochemical staining of angiopoietin-like protein8 in vascular tissue of mice in the group perfused with physiological saline, fig. 4-B is a graph showing immunohistochemical staining of angiopoietin-like protein8 in vascular tissue of mice in the group perfused with angiotensin II, and fig. 4-C is a graph showing a comparison of immunohistochemical staining of angiopoietin-like protein8 in vascular tissue of mice in the above two groups, wherein the horizontal axis shows the group, the vertical axis shows the staining area of angiopoietin-like protein8, the horizontal line at the top of the bar indicates the standard deviation of the staining area of angiopoietin-like protein8, and the uppermost horizontal line and the asterisk in the graph show that the two groups covered with the horizontal line are statistically significant.

As can be seen from fig. 4, the staining area of angiopoietin-like protein8 in the vascular tissue of the mice in the angiotensin II perfused group was significantly larger than that of the saline perfused group, i.e., the expression of angiopoietin-like protein8 in the vascular tissue of the mice with hypertension was significantly larger than that of the normal mice.

Co-localized staining was performed on vascular tissue sections of any of the mice with hypertension in the angiotensin II infused group, i.e., the sections were removed, dried at room temperature for 10min, fixed with 4% paraformaldehyde for 30min, washed 3 times with Phosphate Buffered Saline (PBS), 5min for each wash, incubated with endogenous peroxidase inhibitor for 20min, washed 3 times with PBS for 5min for each wash, serum was blocked for 30min, and dried serum was aspirated and then incubated at 4 ℃ overnight (angiopoietin-like protein8 primary antibody was mixed with a-SMA primary antibody 1:1, diluted with PBS 1: 200, ANGPTL8 primary antibody was purchased from Abcam, and a-SMA primary antibody was purchased from ZSGB-BIO). The next day, after air-drying at room temperature for 30min, the corresponding secondary antibody was incubated for one hour, DIPA mounted, stored at 4 ℃ for 1h, observed by an upright microscope (Nikon, Tokyo, Japan) and images were collected, yielding fig. 5.

As shown in FIG. 5, FIG. 5-A is a staining pattern of angiopoietin-like protein8 in vascular tissue of hypertensive mice, wherein green dotted portions represent angiopoietin-like protein8, FIG. 5-B is a staining pattern of marker protein α -SMA of smooth muscle cells in vascular tissue of hypertensive mice, wherein orange dotted portions represent marker protein α -SMA of smooth muscle cells, FIG. 5-C is a staining pattern of nuclei of smooth muscle cells in vascular tissue of hypertensive mice, wherein blue dotted portions represent nuclei of smooth muscle cells, FIG. 5-D is a co-localized staining pattern of angiopoietin-like protein8, marker protein α -SMA of smooth muscle cells and nuclei of smooth muscle cells in vascular tissue of hypertensive mice, wherein yellow dotted portions are overlapping portions of angiopoietin-like protein8 and marker protein α -SMA of smooth muscle cells due to yellow color obtained by superposition of green and orange colors, and it can be seen that there is also more overlap of the yellow dot with the blue dot. Therefore, in the vascular tissues of hypertensive mice, angiopoietin-like protein8 is expressed at multiple identical locations with smooth muscle cells, and angiopoietin-like protein8 is co-localized with smooth muscle cells.

In the mice perfused with angiotensin II, the stimulation of angiotensin II can cause the formation of hypertension, and further cause the hypertensive vascular remodeling of the mice, and it can be seen from the above experiment that the expression of angiopoietin-like protein8 in the vascular tissue of the mice is significantly increased in the process of producing hypertension and vascular remodeling, that is, angiopoietin-like protein8 is activated in the process of vascular remodeling and formation of hypertension, and angiopoietin-like protein8 is co-localized with smooth muscle cells in the vascular tissue.

Third, angiogenesis-like protein8 and the occurrence and development of mouse hypertension vascular remodeling

Four experimental groups of a first control group, a first perfusion group, a second control group and a second perfusion group having the same number of mice were set, respectively.

The first control group was a C57BL/6 mouse control group, in which 8-10 week-old C57BL/6 mice were perfused with saline using a micro pump and infused continuously for 14 days.

The first infusion group was the C57BL/6 mouse angiotensin II infusion group, in which 8-10 week old C57BL/6 mice were infused with angiotensin II using a minipump at a dose of 490ng/kg/min for 14 days, using angiotensin II purchased from Sigma.

The second control group is a control group of mice inhibiting angiopoietin-like protein8, 4-6 weeks old C57BL/6 mice are injected with serotype 9 adeno-associated virus (AAV-9, purchased from Shanghai Jikai gene) targeting Sh-RNA sequence inhibiting angiopoietin-like protein8 via caudal vein, the injection dose is 2E +10, and normal diet is fed for 4 weeks and then perfused with physiological saline by a micro-pump and continuously infused for 14 days.

The second perfusion group is a mouse angiotensin II perfusion group for inhibiting angiopoietin-like protein8, C57BL/6 mice with the age of 4-6 weeks are injected into caudal vein of serotype 9 adeno-associated virus (AAV-9, purchased from Kjekay gene in Shanghai) targeting Sh-RNA sequence for inhibiting angiopoietin-like protein8, the injection dose is 2E +10, angiotensin II is perfused by a micro pump after feeding for 4 weeks on a common diet, the dose is 490ng/kg/min, and the continuous infusion is carried out for 14 days, and the angiotensin II is purchased from Sigma company.

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-a is a graph showing the change in Systolic Blood Pressure (SBP) of mice in each group in the time period before and after the infusion of angiotensin II or physiological saline, in which the horizontal axis indicates days and the vertical axis indicates systolic blood pressure, the circle indicates the second infusion group, the square indicates the first infusion group, the regular triangle indicates the second control group, and the inverted triangle indicates the first control group, and fig. 6-B is a graph showing the change in Diastolic Blood Pressure (DBP) of mice in each group in the time period before and after the infusion of angiotensin II or physiological saline, in which the horizontal axis indicates days and the vertical axis indicates diastolic blood pressure, the circle indicates the second infusion group, the square indicates the first infusion group, the regular triangle indicates the second control group, and the inverted triangle indicates the first control group.

As can be seen in fig. 6, the mice in the first and second infusion groups both sharply increased diastolic and systolic blood pressure after infusion of angiotensin II, indicating that infusion and stimulation of angiotensin II can lead to the development of hypertension. In 14 days after the mice of each group are subjected to angiotensin II or normal saline, the diastolic pressure and the systolic pressure have no obvious fluctuation, the values of the diastolic pressure and the systolic pressure of the mice of the first perfusion group are the highest, the values of the diastolic pressure and the systolic pressure of the mice of the second perfusion group are the second control group, and the values of the diastolic pressure and the systolic pressure of the mice of the first control group are the lowest. It can be shown that inhibiting angiopoietin-like protein8 can effectively lower the diastolic and systolic blood pressure in mice, i.e. inhibiting angiopoietin-like protein8 can effectively lower blood pressure.

After each group of mice was anesthetized, the mice were vascular perfused with heparin saline to remove residual blood from the blood vessels of the mice. The mouse heart and the proximal aortic tissue were placed in 4% paraformaldehyde overnight at 4 ℃, then transferred to a 30% sucrose solution overnight at 4 ℃, the vascular tissue was taken out of the sucrose solution, blotted, embedded with OCT, and cryosectioned. A plurality of continuous 7-micron-thick frozen sections of each group of mice from the middle of the ventricle to the aortic arch were collected and stored at-20 ℃.

Performing HE staining on the vascular tissue of each group of mice respectively, namely taking out vascular tissue sections of each group of mice, airing at room temperature for 10min, fixing for 30min with 4% paraformaldehyde, washing for 3 times with PBS (5 min each time), staining for 10-15s with hematoxylin, and performing blue reversal with flowing water for 3 min; eosin staining for 10-15s, and 80% ethanol toning until the red is clearly visible; 100% ethanol for 3min, xylene for 5min, air dried in fume hood, neutral gum mounted, observed by upright microscope (Nikon, Tokyo, Japan) and image collected, yielding figure 7.

As shown in fig. 7, fig. 7-a is a graph showing HE staining of vascular tissue of mice of the first control group, fig. 7-B is a graph showing HE staining of vascular tissue of mice of the second control group, fig. 7-C is a graph showing HE staining of vascular tissue of mice of the first infusion group, fig. 7-D is a graph showing HE staining of vascular tissue of mice of the second infusion group, in which purple portions each represent vascular wall of the mice, and fig. 7-E is a graph showing a cylindrical comparison of vascular wall thickness of the mice of each group, in which horizontal axis represents group, vertical axis represents wall thickness (μm), horizontal line at top of the cylindrical shape represents standard deviation of vascular wall thickness of the mice, and the uppermost horizontal line and asterisk in the graph indicate that the two groups covered by the horizontal line are compared and have statistical significance.

As can be seen from fig. 7, the thicknesses of the vascular walls of the mice in the first control group and the second control group are not significantly different, and the thicknesses of the vascular walls of the mice in the first perfusion group and the second perfusion group are both significantly greater than those of the mice in the first control group and the second control group, wherein the thickness of the vascular wall of the blood vessel of the mice in the first perfusion group is greater than that of the blood vessel of the mice in the second perfusion group, which indicates that angiotensin II can cause the thickening of the vascular wall of the blood vessel of the mice, i.e., the blood pressure of the mice is increased due to the perfusion of angiotensin II, and the vascular structure and function of the mice are adaptively changed under a long-term hypertension environment, so that the vascular wall is thickened and the blood vessel is reshaped, and the angiopoietin-like protein8 can effectively reduce the thickness of the vascular wall of the blood.

The method is characterized in that the vascular tissues of each group of mice are respectively dyed with collagen fibers, wherein a kit used for dyeing the collagen fibers is purchased from Sigma company, and the specific steps of dyeing the collagen fibers comprise: (1) taking out vascular tissue slices of each group of mice, airing at room temperature for 10min, fixing with 4% paraformaldehyde for 30min, washing with PBS for 3 times, and oxidizing with 1% potassium permanganate for slicing for 5min (2) every time; (3) washing with water, and bleaching with oxalic acid for 1 min; (4) washing with water, washing with distilled water, and washing with celestite blue dye for 5 min; (5) dropping off residual liquid without washing with water, and dripping Mayer hematoxylin for 3-5 min; (6) flushing with running water for 5-10 min; (7) dyeing the Lichun red bitter acid saturated solution for 5 min; (8) washing with 1% acetic acid aqueous solution; (9) 1% phosphomolybdic acid differentiated section for about 5 min; (10) washing with distilled water; (11) 1% light green or toluidine blue drop-staining for 30 s; (12) washing the slices with 1% acetic acid aqueous solution; (13) differentiating by 95% alcohol, and dehydrating by absolute alcohol; (14) xylene was transparent, the neutral gum was mounted, and observed by an upright microscope (Nikon, Tokyo, Japan) and an image was collected, resulting in fig. 8.

As shown in FIG. 8, FIG. 8-A is a staining pattern of collagen fibers in vascular tissue of the first control group of mice, FIG. 8-B is a staining pattern of vascular tissue collagen fibers of a second control group mouse, FIG. 8-C is a staining pattern of vascular tissue collagen fibers of a first perfused group mouse, FIG. 8-D is a staining pattern of vascular tissue collagen fibers of a second perfused group mouse, wherein the blue-colored clumps represent collagen fibers, the red-colored clumps represent muscle fibers, FIG. 8-E is a comparison of collagen deposition bars for each group of mice, wherein the horizontal axis represents the group, the vertical axis represents the collagen deposition area (%) in the mouse vascular tissue, the horizontal line of the columnar apex represents the standard deviation of the collagen deposition area in the mouse vascular tissue, the top horizontal line and asterisk in the figure indicate that the comparison of the two groups covered by the horizontal line is statistically significant.

As can be seen from fig. 8, there is no significant difference in the collagen deposition area in the vascular tissues of the mice in the first control group and the second control group, and the collagen deposition area in the vascular tissues of the mice in the first perfusion group and the second perfusion group is both significantly larger than that in the first control group and the second control group, wherein the collagen deposition area in the vascular tissues of the mice in the first perfusion group is larger than that in the second perfusion group, which indicates that angiotensin II can cause or promote collagen deposition in the vascular tissues of the mice, while inhibiting angiopoietin-like protein8 can effectively reduce collagen deposition in the vascular tissues of the mice, collagen deposition is one of the markers of vascular remodeling, and the more collagen deposition, the more vascular remodeling, and inhibiting angiopoietin-like protein8 can effectively inhibit collagen deposition in the vascular tissues, i.e., can effectively relieve and inhibit vascular remodeling, thereby reducing blood pressure.

The changes in vascular tissue pathways of the first perfused group and the second control group were analyzed using RNA-seq technology, and verified by q-PCR, and statistically analyzed by prism5.0(GraphPad Software, San Diego, CA), to obtain fig. 9.

As shown in fig. 9, fig. 9-a is a histogram comparing the levels of mRNA related to the endoplasmic reticulum stress factor in mouse vascular tissues, in which all of the endoplasmic reticulum kinase (PERK), elF-2 α, CHOP, activated transcription factor 6(ATF6), and activated transcription factor 4(ATF4) are endoplasmic reticulum stress factors, the mRNA level of the endoplasmic reticulum kinase in mouse vascular tissues of the first perfusion group is 1, the mRNA levels of other groups and each endoplasmic reticulum stress factor are referred to, the horizontal axis represents the group, the vertical axis represents the related mRNA level, the horizontal line at the top of the histogram represents the standard deviation of the levels of mRNA related to the endoplasmic reticulum stress factor in mouse vascular tissues, and the uppermost horizontal line and asterisk in the graph represent that the comparison between the two groups covered by the horizontal line has statistical significance. FIG. 9-B is a histogram comparing the levels of mRNA associated with ER stress factors in mouse vascular tissues, in which each of glucose regulatory protein 94(Grp-94) and G protein-coupled receptor 78(Gpr-78) is an ER stress factor, the mRNA level of glucose regulatory protein 94 in mouse vascular tissues in the first perfusion group is 1, the mRNA levels of the other groups and the ER stress factors are referred to as the mean, the horizontal axis represents the group, the vertical axis represents the level of the mRNA associated with the ER stress factor, the horizontal line at the top of the histogram represents the standard deviation of the mRNA levels associated with the ER stress factor in mouse vascular tissues, and the uppermost horizontal line and the asterisk in the histogram represent the comparison between the two groups covered by the horizontal line, and thus it has statistical significance.

As can be seen from fig. 9, the mRNA levels of various endoplasmic reticulum stress factors of the mice in the first perfusion group were significantly higher than those in the second treatment group, so angiotensin II increased the expression of the endoplasmic reticulum stress factor, while inhibition of angiopoietin-like protein8 effectively decreased the expression of the endoplasmic reticulum stress factor.

Endoplasmic reticulum stress plays an important role in the formation process of hypertension, and inhibition of angiopoietin-like protein8 can reduce the expression of endoplasmic reticulum stress factors and inhibit endoplasmic reticulum stress pathways in smooth muscle cells, thereby preventing and treating hypertension.

Fourth, inhibit the effects of angiopoietin-like protein8 on human vascular smooth muscle cells

Human vascular smooth muscle cells were purchased from Shanghai Xinyu Biotech Co., China Ltd, cell 1X10⁵And inoculating the cells into a six-well plate at a density of/ml, and dividing the human vascular smooth muscle cells into a blank control group, a 6h stimulation group and a 24h stimulation group, wherein the human vascular smooth muscle cells in the blank control group are not treated, the human vascular smooth muscle cells in the 6h stimulation group are stimulated for 6 hours by adopting a hypertensive disease factor angiotensin II, and the human vascular smooth muscle cells in the 24h stimulation group are stimulated for 24 hours by adopting the hypertensive disease factor angiotensin II.

The content level of angiopoietin-like protein8 in human vascular smooth muscle cells of a blank control group, a 6h stimulation group and a 24h stimulation group is respectively detected by using a Western Blot technology, wherein the detection steps of the Western Blot technology comprise: cleaning the glass plate; preparing glue; electrophoresis; film transferring; sealing; primary antibody incubation, wherein the primary antibody is angiopoietin-like protein8 (purchased from Abcam, usa); incubation secondary antibody (purchased from Abcam, usa); for further details, see the explanation of the term "Western immunoblotting" above, and using ChemiDoc^TMThe Touch imaging system (Bio-Rad) imaged, resulting in FIG. 10.

As shown in fig. 10, fig. 10-a is a western-blot diagram of the levels of the content of angiopoietin-like protein8 in human vascular smooth muscle cells of the blank control group, the 6h stimulation group, and the 24h stimulation group, wherein GAPDH is an internal reference of glyceraldehyde-3-phosphate dehydrogenase, black areas indicate the content of angiopoietin-like protein8 and glyceraldehyde-3-phosphate dehydrogenase in each group, and the larger the black areas, the more the content of angiopoietin-like protein8 and glyceraldehyde-3-phosphate dehydrogenase in human vascular smooth muscle cells, and KD is atomic mass unit. FIG. 10-B is a bar graph comparing the levels of angiopoietin-like protein8 in human vascular smooth muscle cells of the placebo, 6h and 24h stimulated groups, in which the horizontal axis represents the groups, the vertical axis represents the levels of angiopoietin-like protein8 (with glyceraldehyde-3-phosphate dehydrogenase as a reference), the horizontal line at the top of the bar represents the standard deviation of the levels of angiopoietin-like protein8 in human vascular smooth muscle cells, and the top horizontal line and the asterisk in the graph represent the comparison between the two groups covered by the horizontal line, which is statistically significant.

As can be seen from fig. 10, the level of angiopoietin-like protein8 in human vascular smooth muscle cells after being stimulated by angiotensin II increases with time, indicating that angiotensin II can increase the expression of angiopoietin-like protein8 in human vascular smooth muscle cells in a time-dependent manner.

Detecting the change of Proliferating Cell Nuclear Antigen (PCNA) in human vascular smooth muscle cells of a blank control group, a 6h stimulation group and a 24h stimulation group respectively by adopting a Western Blot technology, wherein the detection step of the Western Blot technology comprises the following steps: cleaning the glass plate; preparing glue; electrophoresis; film transferring; sealing; primary antibody incubation, wherein the primary antibody is PCNA; incubation secondary antibody (purchased from Abcam, usa); for further details, see the explanation of the term "Western immunoblotting" above, and using ChemiDoc^TMThe Touch imaging system (Bio-Rad) imaged, resulting in FIG. 11.

As shown in fig. 11, fig. 11-a is a western-blot diagram of the level of the proliferating cell nuclear antigen in human vascular smooth muscle cells of the blank control group, the 6h stimulation group, and the 24h stimulation group, GAPDH is an internal reference, glyceraldehyde-3-phosphate dehydrogenase, and black areas indicate the content of proliferating cell nuclear antigen and glyceraldehyde-3-phosphate dehydrogenase in each group, and the larger the black areas, the more the content of proliferating cell nuclear antigen and glyceraldehyde-3-phosphate dehydrogenase in human vascular smooth muscle cells, and KD is atomic mass unit. FIG. 11-B is a bar graph comparing the levels of proliferating cell nuclear antigen in human vascular smooth muscle cells of the blank control group, the 6h stimulated group and the 24h stimulated group, in which the horizontal axis represents the group, the vertical axis represents the level of proliferating cell nuclear antigen (with glyceraldehyde-3-phosphate dehydrogenase as a reference), the horizontal line at the top of the bar graph represents the standard deviation of the level of proliferating cell nuclear antigen in human vascular smooth muscle cells, and the uppermost horizontal line and the asterisk in the graph represent the comparison between the two groups covered by the horizontal line, and are statistically significant.

As can be seen from fig. 11, after human vascular smooth muscle cells are stimulated by angiopoietin-like protein8, the level of proliferating cell nuclear antigen increases with time, which indicates that angiopoietin-like protein8 can stimulate proliferation of smooth muscle cells, and conversely, inhibiting angiopoietin-like protein8 can inhibit proliferation and migration of human vascular smooth muscle cells, thereby delaying hypertension and vascular remodeling caused by hypertension.

All the experiments show that, for patients with hypertension and vascular remodeling related diseases, the expression level of angiopoietin-like protein8 in serum is remarkably increased, and the angiopoietin-like protein8 is inhibited, so that the collagen deposition in smooth muscle cells can be slowed down, the endoplasmic reticulum stress pathway of the smooth muscle cells is inhibited, the proliferation and migration of the smooth muscle cells are inhibited, the blood pressure can be effectively reduced, the thickness of the vascular wall can be effectively reduced, the hypertension and the vascular remodeling related diseases of the hypertension can be effectively prevented and treated, substances inhibiting the angiopoietin-like protein8 are applied to products for preventing or treating the hypertension and/or the vascular remodeling related diseases of the hypertension, the treatment effect of the hypertension and the vascular remodeling related diseases of the hypertension can be effectively improved, the application prospect is good, and the substances inhibiting the angiopoietin-like protein8 and the medicines for treating the hypertension or the vascular remodeling related diseases are applied to the prevention or treatment of the hypertension and the vascular remodeling related diseases of the hypertension together Or in the products of the diseases related to the hypertension vascular remodeling, the traditional treatment effect can be further improved, and the prevention or treatment effect on the hypertension and the diseases related to the vascular remodeling is obviously improved.

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 hypertension and/or diseases related to vascular remodeling is provided.

2. The use according to claim 1, wherein the product for preventing or treating vascular remodeling is a product for preventing or treating a hypertensive vascular remodeling-associated disease.

3. Use according to claim 2, wherein the substance that inhibits angiopoietin-like protein8 prevents or treats hypertension and/or diseases associated with hypertensive vascular remodeling by inhibiting collagen deposition, inhibiting endoplasmic reticulum stress, inhibiting vascular smooth muscle cell proliferation migration.

4. Use according to claim 1, wherein said substance inhibiting angiopoietin-like protein8 is used in combination with other drugs preventing or treating hypertension and/or 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. The 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. Use of a substance inhibiting angiopoietin-like protein8 in the manufacture of a product for inhibiting hypertension and/or vascular remodeling.

9. Use of a substance inhibiting the expression of angiopoietin-like protein8 in the manufacture of a product for inhibiting hypertension and/or vascular remodeling.

10. Application of substance for knocking down or knocking out expression gene of angiopoietin-like protein8 in preparing product formed by hypertension and/or vascular remodeling is disclosed.