CN113398247B - Application of substance for promoting CD44 level in preparation of product for treating/preventing vascular endothelial inflammation by promoting CD44 - Google Patents

Abstract

The application discloses application of a substance for promoting CD44 level in preparing a product for treating/preventing vascular endothelial inflammation by promoting CD 44. The application provided by the application inhibits the generation and development of vascular endothelial inflammation by promoting the CD44 level in a patient with vascular dermatitis, improves the condition of vascular endothelium of the patient, and has very important significance for reducing the morbidity of cardiovascular diseases, improving the prognosis of the patient, reducing the fatality rate of the patient and reducing the national medical cost.

Description

Application of substance for promoting CD44 level in preparation of product for treating/preventing vascular endothelial inflammation by promoting CD44

Technical Field

The present application relates to the field of biology, and more specifically to the use of agents that promote CD44 levels in the manufacture of a product for the treatment/prevention of vascular endothelial inflammation through promotion of CD44 levels.

Background

Vascular endothelial cells are a layer of monocytes that intervene between the vessel wall and the tissue of the bloodstream and act as a physical barrier separating the circulating blood from the vessel wall. Vascular endothelial cells serve as a physiological barrier, and the endothelial structure and function are relatively stable within a certain range; exogenous harmful stimuli (such as long-term hyperglycemia, hypertension, hyperlipidemia and the like) can cause a large amount of inflammatory cytokines to be generated in blood, and under the stimulation of the cytokines, vascular endothelium generates inflammatory reaction, and the function and the structure of the vascular endothelium are changed obviously.

At present, a plurality of medicines for treating vascular endothelial injury exist clinically, such as metformin medicines, insulin, sulfonylurea medicines and the like, but the control rate is still poor, and the incidence rate is increased year by year. Therefore, the early search of a target for preventing or intervening the process and reversing the reaction of the vascular dermatitis has very important significance for reducing the morbidity of cardiovascular diseases, improving the prognosis of patients, reducing the fatality rate of the patients and reducing the national medical cost.

Disclosure of Invention

In order to solve the problems in the prior art, the application provides the application of a substance for promoting the level of CD44 in preparing a product for treating/preventing vascular endothelial inflammation by promoting CD 44.

According to a first aspect of the present application, there is provided a use of an agent promoting CD44 level for the preparation of a product for treating/preventing vascular endothelial inflammation by promoting CD44

In one embodiment of the present application, wherein the substance promoting CD44 levels is used in combination with other drugs for treatment/prevention of vascular endothelial inflammation in said product.

In one embodiment of the present application, wherein the agent that promotes CD44 levels is a CD44 promoter.

In one embodiment of the present application, wherein the substance promoting the level of CD44 is a CD44 recombinant protein.

In one embodiment of the application, the substance promoting the level of CD44 promotes the expression of downstream target gene CD44 in a mode of promoting APPL1 to enter the nucleus and activating transcription factor TCF/LEF.

In one embodiment of the present application, the product for treating/preventing vascular endothelial inflammation further comprises an adjuvant.

In one embodiment of the present application, wherein the product for treating/preventing vascular endothelial inflammation is an injection preparation, an oral tablet or an oral capsule.

In one embodiment of the present application, wherein the inflammation of the vascular endothelium is caused by hypertension, hyperglycemia or hyperlipidemia.

The application of the substance for promoting the level of CD44 in preparing the product for treating/preventing the vascular endothelial inflammation inhibits the generation and development of the vascular endothelial inflammation by promoting the level of CD44 in the body of a patient with vascular dermatitis, thereby achieving the purpose of treating/preventing the vascular endothelial inflammation.

Further features of the present application and advantages thereof will become apparent from the following detailed description of exemplary embodiments thereof, which is to be read in connection with the accompanying drawings.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of the specification, illustrate embodiments of the application and together with the description, serve to explain the principles of the application.

FIG. 1 is a graph comparing the levels of CD44 in the serum of patients in the observation group and subjects in the normal control group.

FIG. 2 is a graph comparing the level of CD44 in HUVEC in a normal medium control group and a high-sugar, high-fat medium-stimulated group.

FIG. 3 is a bar graph comparing the mRNA level of CD44 in HUVEC of a normal medium control group and a high-sugar, high-fat medium-stimulated group.

Fig. 4 is a graph comparing the vascular tissue CD44 levels in mice in the normal diet-fed group and the high fat diet-fed group.

FIG. 5 is a graph showing the comparison of the expression levels of the vascular tissue inflammatory factor ICAM-1 protein in mice fed with high-sugar and high-fat control and mice fed with high-sugar and high-fat stimulation.

FIG. 6 is a graph showing the comparison of the mRNA level of the vascular tissue inflammatory factor ICAM-1 in mice fed with high-sugar and high-fat control and mice fed with high-sugar and high-fat stimulation.

FIG. 7 is a graph comparing the expression levels of p65 protein phosphorylated in the classical inflammatory pathway in mice fed with high-sugar and high-fat control and mice fed with high-sugar and high-fat stimulation.

FIG. 8 is a graph showing a comparison of the levels of TNF-. alpha.mRNA, an inflammatory factor, in mice fed with high-sugar and high-fat control and mice fed with high-sugar and high-fat stimulation.

FIG. 9 is a graph showing the comparison of the level of NLRP3 mRNA in mice fed with high-glucose and high-lipid control and stimulation.

FIG. 10 is a bar graph comparing body weights of six model mice.

FIG. 11 is a bar graph comparing blood glucose of six model mice.

FIG. 12 is a diagram of RNA-seq analysis of differentially expressed gene Pathway analysis.

Figure 13 is an analytical graph of inflammatory molecule enrichment.

FIG. 14 is a graph of the expression of four inflammatory factors in vascular tissue.

FIG. 15 is a diagram showing the expression of ICAM-1 protein in mouse vascular tissues.

FIG. 16 is a Western Blot analysis of human umbilical vein endothelial cells.

FIG. 17 is a laser confocal scan of human umbilical vein endothelial cells.

FIG. 18 is a diagram of RNA-seq technical analysis.

FIG. 19 is a diagram of the human umbilical vein endothelial cell transcription factor activity assay.

FIG. 20 is a graph of mRNA level analysis of human umbilical vein endothelial cell transcription factor.

Figure 21 is a comparison of protein levels of mouse vascular tissue CD 44.

FIG. 22 is a graph comparing the mRNA levels of human umbilical vein endothelial cell inflammatory factor.

FIG. 23 is a Western Blot analysis of mouse vascular tissues.

FIG. 24 is a graph comparing the serum adiponectin levels in the subjects of the observation group and the normal control group.

FIG. 25 is a graph comparing the levels of CD44 in the sera of the observed group of patients and the normal control group of subjects.

FIG. 26 is a graph of a correlation analysis of serum adiponectin and CD44 levels.

Detailed Description

In order to make the application purpose, technical solution and beneficial technical effects of the present application clearer, the present application is described in detail with reference to specific embodiments below. It should be understood that the embodiments described in this specification are only for the purpose of explaining the present application and are not intended to limit the present application.

For the sake of brevity, only some numerical ranges are explicitly disclosed herein. However, any lower limit may be combined with any upper limit to form ranges not explicitly recited; and any lower limit may be combined with any other lower limit to form a range not explicitly recited, and similarly any upper limit may be combined with any other upper limit to form a range not explicitly recited. Further, although not explicitly recited, every point or individual value between endpoints of a range is encompassed within the range. Thus, each point or individual value can form a range not explicitly recited as its own lower or upper limit in combination with any other point or individual value or in combination with other lower or upper limits.

In the description herein, it is to be noted that, unless otherwise specified, "above" and "below" are inclusive and "one or more" means "several" are two or more.

The above summary of the present application is not intended to describe each disclosed embodiment or every implementation of the present application. The following description more particularly exemplifies illustrative embodiments. At various points throughout this application, guidance is provided through a list of embodiments that can be used in various combinations. In each instance, the list is merely a representative group and should not be construed as exhaustive.

CD44 is a non-kinase transmembrane proteoglycan encoded by a gene located on human chromosome 11 or mouse chromosome 2, which is involved in intercellular interactions, cell adhesion and migration. CD44 is a receptor for hyaluronic acid and can also interact with other ligands, such as osteopontin, collagen and Matrix Metalloproteinases (MMPs). CD44 is expressed in a variety of cell types, and several studies have shown that CD44 can promote tumorigenesis, cancer cell migration, and proliferation. There are also a number of reports on the use of CD44 as a molecular target for cancer therapy to reduce the symptoms and progression of cancer by inhibiting CD44 expression and activity using neutralizing antibodies, polypeptides, drugs, etc.

1. Western immunoblotting (Western blot)

Western immunoblotting is a method in which proteins are 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:

a. cleaning glass sheets

The glass plates are divided into 1.5mm and 1.0mm, 1.5mm being conventionally used (the comb teeth have corresponding 1.5 and 1.0, 15 and 10 holes, respectively).

Firstly, cleaning with clear water, then scrubbing with a detergent, then continuously cleaning with clear water, standing after the clear water is cleaned, cleaning with distilled water, and fixing the large plate and the small plate on a glue preparation underframe.

b. Glue preparation

The separating gel formula comprises: 10% of the gels are suitable for molecules with a high molecular weight, and 12% of the gels are suitable for molecules with a low molecular weight (10-60).

The concentrated gel formula comprises:

preparing separation gel, adding other components in the preparation process of the separation gel, adding TEMED, mixing well, rapidly adding into a 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.

c. 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 marker: 3/5/7/10u (the method of distinguishing left and right can be selected flexibly according to the sample loading condition, and different amounts of marker are added on left and right to distinguish left and right).

ii. And (3) determining the sample loading amount: 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.

d. 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, double distilled water: methanol: 10 × electrotransfer solution was 7:2:1(700ml, 200ml, 100 ml).

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, and after the small glass plate is removed, the glue is slightly proper in the upper part, the lower part and the left part and the right part. Cut off a little to avoid scraping the separating glue, and carefully peel off the separating glue 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.

e. 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.

f. Primary anti-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 ℃.

g. 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.

2、Real-time 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) with a narrow concept means that the purpose 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 (a fluorescent hybridization probe ensures specificity), and meanwhile, false negative results are effectively eliminated (the amplification efficiency is zero) by using an internal control.

3. Small interfering RNA transfection

24h before transfection, HUVEC cells were digested with 0.25% trypsin and plated in 6-well plates, and when cells grew to 70% -80% confluence, they were replaced with 1% fetal bovine serum-containing DMEM medium, 1ml per well. Mu.l of 25. mu.M siRNA was added to 100. mu.l of DMEM serum-free medium and mixed gently. Mu.l of siPort transfection reagents were diluted with 100. mu.l of serum-free DMEM, gently mixed, and left at room temperature for 10 minutes. Mixing the diluted siRNA and siPort Transfection Reagents; gently mixed and left at room temperature for 10 minutes. 200 μ l of siRNA/siPort Transfection Reagents complex was added to a 6-well plate and the cell culture plate was gently shaken back and forth. After the cells were incubated at 37 ℃ for 6-8h in an incubator, the endothelial cell medium containing 10% fetal bovine serum was replaced, and the cells were processed and collected after 72 h.

4. Transcription factor activation assay plate array II

Transcription factor activation assay plate array II (purchased from signalis) was used to analyze the activity of transcription factors in HUVEC cells according to the manufacturer's instructions. Nuclear proteins were isolated from HUVEC cells using a nuclear extraction kit (purchased from Signosis, Sunnyvale, Calif.). Biotin-labeled probes based on consensus sequences of transcription factor DNA binding sites were mixed with 15. mu.g of nuclear protein extract to form transcription factor/probe complexes. The bound probe is separated from the complex and hybridized to a plate coated with a sequence that is previously complementary to the probe. The captured DNA probe was detected with streptavidin-HRP and the signal intensity was measured with a microplate luminometer. Relative gene expression levels were calculated using the CT method and normalized to the average expression level of five housekeeping genes.

5. Transcriptome sequencing analysis (RNA-seq)

Total RNA was isolated from rat aortic tissue by the TRIzol method (purchased from Invitrogen). The mRNA is reverse transcribed into double-stranded cDNA fragments. mRNA libraries were generated from NEB Next Ultra direct RNA library preparation kit for Illumina (# E7530L, NEB, Ispawich, USA) according to the manufacturer's protocol. Library quality control was performed and then the RNA library was sequenced at 10pM on Illumina HiSeq 4000 to generate 150bp paired-end reads. RNA-seq differential expression analysis was performed by DESeq2 v1.20.0 (http:// bioconductor. org/packs/release/bioc/html/DESeq 2. html). After RNA-seq differential expression analysis was performed, functional annotation was performed by GO or Pathway enrichment analysis (Gene Ontology, http:// geneontology. org /). GO analysis or Pahway analysis with FDR <0.05 was considered significantly enriched.

6. Human transcription factor detection kit

mRNA levels of Wnt/β -catenin pathway transcription factors in HUVEC cells were analyzed using human transcription factors (purchased from Qiagen, USA) according to the manufacturer's instructions. Total RNA was isolated by Trizol reagent method (Invitrogen) for cDNA synthesis. qRT-PCR was performed on a 7500Real-Time PC system (Thermo Fisher Scientific, Inc) using RT2 SYBR Green Mastermix (PARN-026Z, QIAGEN). The PCR conditions included: denaturation at 95 ℃ for 10min, 95 ℃ for 15 sec, 40 cycles and 60 ℃ for 1 min. The CT values were used to calculate gene expression levels. Fold change in expression was determined by 2- Δ Δ CT.

For convenience of explanation, the relevant abbreviations and english words used in this embodiment are explained as follows:

WT: wild control mice; APPL1 KO: apl 1 gene knock-out mice; ND: common feed; HFD: high fat and high sugar feed; APN: adiponectin; NG/NL: a common culture medium; HG/HL: a high-fat high-sugar culture medium; enrichment: enriching a passage; vehicle physiological saline perfusate group; siAPPL1 knocking down APPL1 gene; siCD44 knock-down of CD44 gene.

The present application provides specific uses of the substance that promotes CD44 levels described below.

The application provides an application of a substance promoting CD44 level in preparing a product for treating/preventing vascular endothelial inflammation by promoting CD44 level. The substance that promotes CD44 levels in the manufactured product or medicament treats vascular dermatitis in a patient by promoting CD44 levels in the patient.

In one embodiment of the present application, the inflammation of the vascular endothelium is caused by hypertension, hyperglycemia or hyperlipidemia. For example, the hyperglycemic vascular inflammatory response refers to the adaptive alteration of the vascular structure in a long-term hyperglycemic environment. It is found that the hyperglycemia vascular inflammation reaction is mainly shown by inflammatory cell adhesion, migration and chemotaxis, vasodilation, increase of vascular permeability and the like. It is understood that vascular dermatitis has similar responses and pathologies to those skilled in the art, and thus, vascular dermatitis may also be caused by, or as a complication of, other diseases.

In one embodiment of the present application, the substance that promotes the level of CD44 may be a promoter that promotes the expression level of CD44 in vivo. For example, the promoter may be a CD44 recombinant protein, or other drug that can promote expression of CD44 in vivo.

In one embodiment of the present application, the product for treating/preventing vascular endothelial inflammation may be in the form of injection, oral tablet, or oral capsule, which are conventional formulations known to those skilled in the art.

In one embodiment of the present application, the substance that promotes CD44 levels is used in combination with other drugs for the treatment/prevention of vascular endothelial inflammation in the product. Although the present application does not provide experiments related to the combined use of the drug and other drugs for treating/preventing vascular endothelial inflammation, it will be apparent to those skilled in the art that the substance for promoting the CD44 level can achieve the purpose of treating/preventing vascular endothelial inflammation as long as it can promote the expression of CD44 level in the body of a patient.

For example, the other drugs for treating/preventing vascular endothelial inflammation may be drugs having a certain therapeutic effect on inflammatory factors, such as anti-inflammatory drugs, which may be pharmaceutical compounds, pharmaceutical compositions, and the like, or may be drugs in various dosage forms, such as tablets, granules, powders, capsules, injections, and the like, which is not limited in this application.

The product for treating/preventing vascular endothelial inflammation further comprises auxiliary materials, and on the basis of the application of the substance for promoting the expression of the CD44 level in the preparation of the product for treating/preventing vascular endothelial inflammation by promoting the CD44 level, the reasonable selection of the appropriate auxiliary materials is routine for those skilled in the art and is not specifically described herein.

In one embodiment of the present application, the substance that promotes the level of CD44 promotes the expression of downstream target gene CD44 in a manner that promotes the nuclear entry of APPL1 and the activation of the transcription factor TCF/LEF. Finding a therapeutic target is very important for treating/preventing vascular endothelial inflammation, and substances such as adiponectin at the level of CD44 promote the entry of APPL1 into the nucleus, activate transcription factors TCF/LEF and promote the expression of downstream target genes CD44 so as to inhibit the generation or development of inflammation.

APPL1(adaptor protein, PH domain and leucoine linker binding 1) is an adaptor protein consisting of 709 amino acids, including the BAR domain, PH domain and carboxy-terminal PTB domain of amino acids.

The application shows that the level of CD44 has a direct relation with vascular endothelial inflammation through various examples, and various inflammatory factors for reacting vascular inflammation, such as TNF-alpha, IL-1 beta, ICAM-1, NLRP3 and the like, can be obviously inhibited by promoting the level of CD44 in vivo. Based on the control experiments and administration experiments of the present invention, it is obvious to those skilled in the art that other substances capable of promoting the level of CD44 in vivo can achieve the effect of treating/preventing vascular endothelial inflammation.

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

Example I patient control experiment

Patients with initial diagnosis of diabetes mellitus are continuously collected at an Anzhen hospital in Beijing, affiliated to the university of capital medical science, according to the Chinese diabetes diagnosis standard: firstly, fasting blood glucose (FPG) is more than or equal to 7.0 mmol; ② for those with typical diabetes symptoms (polyuria, polydipsia and unexplained weight loss), the arbitrary blood sugar is more than or equal to 11.1 mmol/L. Diabetes can be diagnosed by meeting one of the above criteria. The exclusion standard is secondary hypertension such as kidney diseases, pheochromocytoma, primary aldosteronism and the like; ② severe arrhythmia; moderate and severe cardiac insufficiency; fourthly, active liver dysfunction or ALT or AST higher than 3 times of normal upper limit found by physical examination; malignant diseases: a tumor; sixthly, anemia is generated; acute and chronic inflammatory diseases: including rheumatic arthritis, rheumatoid arthritis, etc. 74 diabetic patients were selected as the observation group according to the above screening criteria.

30 subjects were randomly selected as normal control groups from the population who had been subjected to health examination in the same hospital examination center and had normal blood sugar. Collecting multiple routine test indexes such as blood lipid, blood glucose and blood, and carefully inquiring the disease history such as cardiovascular diseases, liver and kidney diseases and family history.

In all the selected cases, 10ml blood samples were taken intravenously after 8-10 hours of fasting, centrifuged at 3000rpm for 10 minutes, and then serum was extracted and frozen in a freezer at-80 ℃.

The serum CD44 content of the observation group patients and the normal control group subjects was measured by a high-sensitivity enzyme-linked immunosorbent assay (ELISA) (kit was purchased from Cat #38E7963A34, Cloud-Clone Corp), respectively.

CD44 in the ELISA kit is coated in 96-well micro-porous plate in advance to form solid phase carrier. The standard and the specimen are added into the micropore, wherein CD44 is combined with the antibody coated on the solid phase carrier, then biotinylated CD44 antibody is added, after washing the unbound biotinylated antibody, horseradish peroxidase (HRP) labeled avidin is added, after washing again, 3',5,5' -tetramethyl benzidine (TMB) substrate is added 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 the concentration of CD44 in the sample. The absorbance (o.d. value) was measured at a wavelength of 450nm with a microplate reader to obtain a standard curve, and then the CD44 concentration in the sample was calculated based on the standard curve and statistically analyzed using SPSS software (IBM corp., Armonk, NY, USA), giving fig. 1.

FIG. 1 is a bar graph comparing the CD44 levels in the sera of patients in the observation group and normal control subjects, wherein the horizontal axis represents the group, the vertical axis represents the CD44 level (ng/ml), the horizontal line above the bar represents the standard deviation of the CD44 level, and the asterisks indicate that the experiment and the results of the experiment are statistically significant. As can be seen, the content of CD44 in the serum of the observed group patients is obviously lower than that of CD44 in the serum of the normal control group subjects, so that the expression level of CD44 in the serum of the diabetic patients is obviously reduced, and CD44 is inhibited in the diabetic patients.

Example two human umbilical vein endothelial cell HUVEC experiment

The human umbilical vein endothelial cell HUVEC (purchased from the national science and technology resource sharing service platform Beijing cooperative cell center) is taken as a model, and the experiment is divided into two groups: common culture medium control group and high-sugar high-fat culture medium stimulation group, placing in 5% CO₂Incubated at 37 ℃ for 72 hours. Common media components include endothelial cell culture medium (purchased from ScienCell, USA) + 10% fetal bovine serum (purchased from Life technologies, Germany) + 1% antibiotic Penicillin-Streptomyces (purchased from Thermo Fisher Scientific, USA); the high-sugar and high-fat medium components comprise 4.5g/L D-glucose medium (from Life technologies, Germany), 5% high-fat working solution (0.01397g sodium palmitate and 1g bovine serum albumin dissolved in 10ml phosphate buffer PBS to make working solution), 10% fetal bovine serum (from Life technologies, Germany), 1% antibiotic Penicillin-Streptomyces (from Thermo Fish, USA)er Scientific)。

After successful cell modeling, cell proteins were extracted by lysing the cells using lysate lyssbuffer (available from Bilun biology, China) and 1% protease inhibitor (available from Thermo Fisher Scientific, USA).

Western Blot technique was used to detect the protein levels of the cell model CD44 in the control group of normal medium and the high-sugar and high-lipid medium-stimulated group, respectively, to obtain FIG. 2.

As can be seen from FIG. 2, the protein level of HUVEC cells CD44 in high-sugar high-lipid culture (HG/HL) was significantly lower than that of CD44 in normal culture (NG/NL) cells.

Respectively detecting the expression level of CD44mRNA in HUVEC of a high-sugar and high-fat culture medium stimulation group and a normal culture medium control group by adopting an RT-PCR technology, wherein the detection steps of the RT-PCR technology comprise: RNA was extracted, reverse transcribed, PCR amplified, and statistically analyzed by Graph prism8.0(GraphPad Software, San Diego, Calif.) to give FIG. 3.

As shown in fig. 3, fig. 3 is a histogram comparing the mRNA levels of CD44 in HUVECs of the normal medium control group and the high-sugar high-fat medium-stimulated group, wherein the mRNA level of CD44 in HUVECs of the normal medium control group is 1, the mRNA level of CD44 in HUVECs of the high-sugar high-fat medium-stimulated group is based on the mRNA level of CD44 in cells of the normal control group, the horizontal axis represents the group, the vertical axis represents the mRNA level of CD44, the horizontal line at the top of the histogram represents the standard deviation of the CD44mRNA level, and the uppermost horizontal line and the asterisk in the graph represent the comparison between the two groups covered by the horizontal lines, and it is statistically significant.

As can be seen from fig. 3, the mRNA level of CD44 in the high-sugar, high-lipid medium-stimulated group was significantly lower than that of CD44 in the normal control group.

EXAMPLE III expression of CD44 in mouse model vascular tissue

1. Mouse model establishment

40 mice (purchased from Beijing sbeful laboratory animals Co., Ltd.) from 8-10 weeks C57BL/6 were selected and divided into 2 groups: the group was fed with 20 animals per group for 10 weeks. Wherein the high fat diet feeding group was high fat diet (purchased from Changzhou rat-rat two-biotechnology limited) containing 60% kcal% of fat, and the diabetes model was induced by diet. The common feed feeding group feeds according to common feed.

2. Dissect

After the model building is successful, carrying out gas anesthesia treatment on mice of each group by using isoflurane, disinfecting and opening the skin of the mice, quickly opening the thoracic cavity of the mice, taking blood from the apex of the heart, fully opening the thoracic cavity and abdominal cavity of the mice after being perfused by normal saline, stripping redundant tissues, fully exposing aorta blood vessels, fully separating tissues around the blood vessels under a body type 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 standby.

3. Western Blot detection

The protein level of CD44 in the vascular tissues of mice in the ordinary feed-fed group and the high-fat feed-fed group was detected by Western Blot technique. The detection steps of the Western Blot technology comprise: cleaning a glass plate, preparing glue, performing electrophoresis, transferring a membrane, sealing, incubating and developing. Wherein the primary antibody is CD44 (available from Cell Signaling Technology, USA); secondary antibody (purchased from Cell Signaling Technology, usa) was incubated. Using ChemiDoc^TMThe Touch imaging system (Bio-Rad) performed imaging, resulting in FIG. 4.

Fig. 4 a is a western-blot diagram of vascular tissue CD44 of mice in normal diet feeding group (ND) and high fat diet feeding group (HFD), wherein GAPDH is glyceraldehyde-3-phosphate dehydrogenase (i.e., internal reference, black area indicates the content of vascular tissue CD44 and glyceraldehyde-3-phosphate dehydrogenase, and the larger black area, the greater the content of CD44 in the vascular tissue of the mice, and dalton (KD) is atomic mass unit. Fig. 4 b is a Graph showing a comparison of the protein level histograms of vascular tissue CD44 of Normal Diet (ND) and High Fat Diet (HFD) mice, which were statistically analyzed using Image Lab Software (Bio-Rad) to plot black areas, and then using Graph Prism8.0 Software (Graph pad Software, San Diego, CA) to plot a histogram, wherein the horizontal axis represents the group classification, the vertical axis represents the protein level of CD44 (with glyceraldehyde-3-phosphate dehydrogenase as a reference), the horizontal line at the top of the histogram represents the standard deviation of the CD44 level, and the uppermost horizontal line and 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. 4, the protein level of CD44 in the vascular tissue of the high-fat-fed mice was significantly lower than that of CD44 in the vascular tissue of the normal control mice.

EXAMPLE four mouse treatment experiment with recombinant protein CD44

1. Mouse model establishment

C57BL/6 mice (purchased from beijing sbeful laboratory animals ltd) high-sugar, high-fat control-fed group: high fat diet (purchased from Changzhou mice, one mouse, two Biotech Co., Ltd.) was fed to C57BL/6 mice for 10 weeks, and high sugar high fat mice were perfused by saline micro-pump (micro-pump purchased from ALZET scientific instruments, USA) at a dose of 0.25ug/g/day and infused continuously by micro-pump for 7 days for a total of 5 mice.

C57BL/6 mice fed group with high-sugar and high-fat stimulation (given CD44 treatment): high fat diet (purchased from Changzhou mouse-two Biotech limited) was fed to C57BL/6 mice for 10 weeks, and CD44 recombinant protein (purchased from Shanghai Biotech, China) was infused into high sugar high fat mice at a dose of 0.25ug/g/day by a micro pump for 7 days, for a total of 5 mice.

2. Dissect

Mice in each group after 4-5 hours fasting were anesthetized with isoflurane gas. After anesthesia, the skin was disinfected and the abdomen was opened, the thoracic cavity of the mouse was opened rapidly, and the apex of the heart was bled to about 1ml and placed in a centrifuge tube. Fully opening the pleuroperitoneal cavity of the mouse after the perfusion of the normal saline, stripping redundant tissues, fully exposing aorta blood vessels, fully separating tissues around the blood vessels under a body type 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 standby. After the material drawing is finished, the whole blood sample of the mouse is centrifuged at 3000rpm for 10 minutes, and then serum is sucked and frozen in a refrigerator at the temperature of minus 80 ℃.

The content of CD44 in the serum of CD 44-treated high-sugar high-fat mice and normal saline-perfused high-sugar high-fat mice was determined by a high-sensitivity enzyme-linked immunosorbent assay (ELISA) (kit from Cat #38E7963A34, Cloud-Clone Corp), respectively. CD44 in the ELISA kit is coated in a 96-well microplate in advance to form a solid phase carrier. The standard and the specimen are added into the micropore, wherein CD44 is combined with the antibody coated on the solid phase carrier, then biotinylated CD44 antibody is added, after washing the unbound biotinylated antibody, horseradish peroxidase (HRP) labeled avidin is added, after washing again, 3',5,5' -tetramethyl benzidine (TMB) substrate is added 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 the concentration of CD44 in the sample. After an enzyme-labeling instrument is used for measuring absorbance (O.D. value) at the wavelength of 450nm to obtain a standard curve, the concentration of CD44 in a sample is calculated according to the standard curve, SPSS software (IBM Corp., Armonk, NY, USA) is used for carrying out statistical analysis, and the result shows that the content of CD44 in the serum of a mouse with high sugar and high fat content treated by CD44 is obviously higher than the content of CD44 in the serum of the mouse with high sugar and high fat content infused by physiological saline, so that the CD44 is successfully supplemented in the mouse through CD44 recombinant protein.

The protein levels of ICAM-1 in vascular tissues of a group of high-sugar and high-fat mice perfused with CD44 and a group of high-sugar and high-fat mice perfused with physiological saline are respectively detected 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 ICAM-1 (purchased from Abcam, usa); secondary incubation antibody (available from Cell Signaling Technology, usa); developing and using ChemiDoc^TMThe Touch imaging system (Bio-Rad) imaged, resulting in FIG. 5.

As can be seen from FIG. 5, ICAM-1 was significantly lower in the protein level in the CD 44-perfused high-sugar, high-lipid mouse treated group (HFD + CD44) than in the normal saline-perfused high-sugar, high-lipid mouse (HFD + Vehicle) vascular tissue CD 44.

Respectively detecting the mRNA expression level of ICAM-1 in the vascular tissues of a group of high-sugar and high-fat mice perfused with CD44 and a group of high-sugar and high-fat mice perfused with physiological saline by adopting an RT-PCR technology, wherein the detection steps of the RT-PCR technology comprise: RNA was extracted, reverse transcribed, PCR amplified, and statistically analyzed by Graph prism8.0(GraphPad Software, San Diego, Calif.) to give FIG. 6.

FIG. 6 is a histogram comparing the mRNA levels of ICAM-1 in vascular tissue of normal saline-and CD 44-perfused high-sugar, high-lipid mice. The mRNA level of a hyperglycemia mouse perfused with physiological saline was 1, the ICAM-1mRNA level of the vascular tissue of a CD44 perfused hyperlipidemia mouse was found to be statistically significant by comparing the two groups covered with the horizontal line, wherein the horizontal axis represents the group, the vertical axis represents the mRNA level of ICAM-1, the horizontal line at the top of the bar represents the standard deviation of the ICAM-1mRNA level, and the upper horizontal line and the asterisk in the figure represent the reference of the ICAM-1mRNA level of a hyperlipidemia mouse perfused with physiological saline.

As can be seen from FIG. 6, the mRNA level of ICAM-1 in vascular tissue of CD 44-perfused high-sugar, high-lipid mice was significantly lower than that of ICAM-1 in vascular tissue of saline-perfused high-sugar, high-lipid mice.

The protein levels of NF-kB (p65) and phosphorylated NF-kB (p-p65) in the vascular tissues of CD 44-perfused high-sugar high-fat mice and normal saline-perfused high-sugar high-fat mice are respectively detected by using a Western Blot technique. The detection steps of the Western Blot technology comprise: cleaning the glass plate; preparing glue; electrophoresis; film transferring; sealing; primary antibody incubation, with primary antibodies NF- κ B (p65) and phosphorylated NF- κ B (p-p65) (both available from Cell Signaling Technology, usa); secondary incubation antibody (available from Cell Signaling Technology, usa); and (6) developing. And using ChemiDoc^TMThe Touch imaging system (Bio-Rad) imaged, resulting in FIG. 7.

FIG. 7A is a western-blot of vascular tissue p-p65 in the group of normal saline-and CD 44-perfused high-sugar and high-fat mice, where p65 is an internal reference, the black area indicates the content of vascular tissues p-p65 and p65, and the larger the black area, the greater the content of p65 in the vascular tissues of the mice, and Dalton (KD) is in atomic mass units. Panel B in figure 7 is a bar graph comparing the blood vessel tissue p-p65 protein levels of a group of normal saline perfused high sugar, high fat mice and CD44 perfused high sugar, high fat mice. The black area size was marked using Image Lab Software (Bio-Rad), then statistically analyzed using Graph Prism8.0 Software (Graph pad Software, San Diego, CA) and plotted in a bar Graph, where the horizontal axis represents the group, the vertical axis represents the protein level of p-p65 (referred to as p65), the horizontal line at the top of the bar Graph represents the standard deviation of the p-p65 level, and the uppermost horizontal line and asterisk in the Graph represent the statistical significance of comparing the two groups covered by the horizontal lines.

As can be seen from FIG. 7, the protein level of p-p65 in the blood vessel tissue of the treated group of high-sugar, high-fat mice perfused with CD44 was significantly lower than that of p-65 in the blood vessel tissue of the high-sugar, high-fat mice perfused with physiological saline.

The expression level of TNF-alpha mRNA in the vascular tissues of the group of CD 44-perfused high-sugar and high-fat mice and the group of normal saline-perfused high-sugar and high-fat mice were measured by RT-PCR technique, respectively, and the results are shown in FIG. 8. The mRNA level of TNF-alpha in the vascular tissue of the high-sugar and high-fat mice perfused with CD44 is obviously lower than that of the vascular tissue of the high-sugar and high-fat mice perfused with physiological saline.

The expression level of NLRP3 mRNA in the blood vessel tissues of the group of CD 44-perfused high-sugar and high-fat mice and the group of normal saline-perfused high-sugar and high-fat mice were respectively detected by RT-PCR technology, and the result is shown in FIG. 9. The mRNA level of NLRP3 of blood vessel tissue of CD44 perfused high-sugar high-fat mouse is obviously lower than that of NLRP3 of blood vessel tissue of normal saline perfused high-sugar high-fat mouse.

EXAMPLE V experiment of adiponectin administration to mice

In this example, unless otherwise specified, each test material used was the same as in the other examples, i.e., the same purchase source, the same trade name, the same composition, etc.

First, APPL1 mediated adiponectin inhibition vascular endothelial inflammation test

1. Group model building

The experiments were divided into six groups:

c57BL/6 mouse control group: the normal diet was fed for 10 weeks, and the normal saline was infused for 2 weeks at a dose of 0.25ug/g/day for 14 days by continuous infusion through a micro pump for a total of 5 mice.

C57BL/6 mice high-sugar and high-fat feeding group: the high-sugar and high-fat feed is fed for 10 weeks, the normal saline is pumped for 2 weeks, the dosage is 0.25ug/g/day, and the infusion is continuously carried out for 14 days through a micro pump, and the total number of the mice is 5.

C57BL/6 mice were fed high-sugar and high-fat and given adiponectin-treated groups: the high-sugar, high-fat diet was fed for 10 weeks at a dose of 0.25ug/g/day of adiponectin (purchased from Peprotech Corp.) and continuously infused by a minipump for 14 days for a total of 5 mice.

APPL 1-/-knockout mouse control group: APPL 1-/-Gene knock-out mice (purchased from Sphachis biosciences, Inc., China) were fed with normal feed for 10 weeks, infused with saline at a dose of 0.25ug/g/day by a micro pump, and continuously infused with the micro pump for 14 days for a total of 5 mice.

APPL 1-/-knockout mice high-fat high-sugar feeding group: APPL 1-/-gene knock-out mice were fed with high-sugar and high-fat diet for 10 weeks, infused with saline micro-pump at a dose of 0.25ug/g/day for 14 days, for a total of 5 mice.

APPL 1-/-mice were fed high-sugar and high-fat and given to the adiponectin-treated group: the high-sugar and high-fat diet was fed for 10 weeks with adiponectin dose of 0.25ug/g/day by continuous infusion through a micropump for 14 days, for a total of 5 mice.

The body weight of each model mouse was weighed using an analytical balance, and fig. 10 is a bar graph comparing the body weights of the 6 model mice described above. The horizontal axis represents the group, the vertical axis represents the weight of the mice, and the left axis represents the weight increase of the C57BL/6 mice fed with high sugar and high fat compared with the C57BL/6 mice control group, and the weight increase is statistically different. # indicates that the C57BL/6 mice were fed high sugar and high fat and the adiponectin treatment group was given a body weight difference between the two groups compared to the C57BL/6 mice fed high sugar and high fat group. And & indicates that there was a difference in body weight between the two groups compared between the APPL 1-/-mouse high sugar and high fat fed and adiponectin-treated group and the C57BL/6 mouse high sugar and high fat fed and adiponectin-treated group.

As can be seen from fig. 10, the mice fed the high-fat and high-sugar diet had increased body weight and Adiponectin (APN) treatment decreased body weight.

2. Dissect

After the successful modeling, the mice in each group are subjected to gas anesthesia treatment by using isoflurane after 4-5 hours of empty belly, after anesthesia, the skin is disinfected and the belly is opened, the chest cavity of the mice is opened rapidly, and the apex of the heart is bled to about 1ml and placed in a centrifuge tube. Fully opening the pleuroperitoneal cavity of the mouse after the perfusion of the normal saline, stripping redundant tissues, fully exposing aorta blood vessels, fully separating tissues around the blood vessels under a body type 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 standby.

3. Test for inhibiting vascular endothelial inflammation

Using a glucose determination kit (purchased from China Zhongsheng North control Biotechnology limited), and uniformly mixing 10ml of glucose oxidase and 90ml of phosphate buffer solution according to the kit specification to obtain a working solution; and (3) performing serial gradient dilution on the standard substance, adding 0.01ml of the standard substance, 0.01ml of the mouse blood sample and 1.5ml of the working solution into a cuvette, uniformly mixing, preserving the temperature at 37 ℃ for 10 minutes, and detecting the absorbance at the wavelength of 505nm by using an enzyme-labeling instrument. FIG. 11 is a bar graph comparing the blood glucose levels of the above 6 model mice, wherein the horizontal axis represents the group, the vertical axis represents the fasting blood glucose level of the mice, and the left axis represents the blood glucose level increase of the C57BL/6 mice fed with high blood glucose and high fat compared with the C57BL/6 mice control group, and the blood glucose level increase is statistically different. # indicates that the group treated with C57BL/6 mice and administered adiponectin had a difference in blood glucose between the two groups compared to the group treated with C57BL/6 mice. And & indicates that blood glucose was different between the two groups compared to the adiponectin-treated group when apl 1-/-mice were fed high-sugar and high-fat and administered to the adiponectin-treated group and the C57BL/6 mice were fed high-sugar and high-fat and administered to the adiponectin-treated group. As can be seen from FIG. 2, the blood glucose of mice fed with high-fat and high-sugar diet was increased, and the blood glucose of mice was decreased by adiponectin treatment.

Analyzing and detecting the change of the vascular tissue inflammation pathway of the C57BL/6 mice after the adiponectin treatment by adopting an RNA-seq technology, wherein the RNA-seq technology comprises the following detection steps: extracting RNA of the vascular tissue; PCR amplification; sequencing; controlling the quality; and (6) data and processing. FIG. 12 is a graph showing the differential expression of gene Pathway analysis in RNA-seq analysis, in which the horizontal axis shows the magnitude of FDR value of the enrichment degree and the vertical axis shows the different pathways, and the larger the FDR value on the horizontal axis, the more significant the differential enrichment. As can be seen from fig. 12, the inflammatory pathways are significantly enriched in the enrichment pathway.

13 differential genes (screened from the enrichment pathway described above) were subjected to mapping analysis using the Pheatmap package under conditions (P ≦ 0.05) using the R language programming technique (version 3.6.0), as shown in FIG. 13, where the icon changes from bottom to top in light gray to dark gray to indicate differential gene expression down-regulation and up-regulation. From left to right, each three boxes represent a group of samples, which are the differential expression of the vascular tissue inflammation related gene of the normal control group, the differential expression of the vascular tissue inflammation related gene of the high-glucose and high-lipid mouse, and the differential expression of the vascular tissue inflammation related gene of the adiponectin-treated high-glucose and high-lipid mouse. As can be seen from fig. 13, the mice fed the high-sugar and high-fat diet promoted vascular inflammatory response in the mice, while adiponectin treatment inhibited vascular inflammatory response.

As the RNA-seq technology is a high-throughput sequencing technology, the result has certain inaccuracy, and the result needs RT-PCR to be verified, thereby proving that the adiponectin inhibits the reaction of the vascular dermatitis.

Respectively detecting the expression conditions of the inflammatory factors of the vascular tissues by adopting an RT-PCR technology, wherein the detection steps of the RT-PCR technology comprise: RNA was extracted, reverse transcribed, PCR amplified, and statistically analyzed by Graph prism8.0(GraphPad Software, San Diego, Calif.).

As shown in FIG. 14, A is a C57BL/6 mouse control group, a C57BL/6 mouse high-sugar high-fat fed group, a C57BL/6 mouse high-sugar high-fat fed group and adiponectin-treated group, and APPL 1-/-mouse high-sugar high-fat fed group and ICAM-1mRNA level of vascular tissue of the group treated with adiponectin, wherein:, it means that ICAM-1mRNA level of the vascular tissue of the C57BL/6 mouse control group is 1 and ICAM-1mRNA level is different between the two groups compared with the C57BL/6 mouse control group in the C57BL/6 mouse high-sugar high-fat fed group. # indicates that the mRNA level of ICAM-1 in vascular tissue of the group fed with high sugar and high fat in C57BL/6 mice was 1 and the mRNA level of ICAM-1 was different between the two groups in the group fed with high sugar and high fat in C57BL/6 mice and administered to adiponectin-treated group compared to the group fed with high sugar and high fat in C57BL/6 mice. And & indicates that mRNA level of ICAM-1 in vascular tissue of C57BL/6 mouse fed with high sugar and administered to adiponectin-treated group was 1, and there was a difference in mRNA level of ICAM-1 between the two groups, compared to those of APPL 1-/-mouse fed with high sugar and administered to adiponectin-treated group and those of C57BL/6 mouse fed with high sugar and administered to adiponectin-treated group. The horizontal axis represents the group, the vertical axis represents the mRNA level of ICAM-1, and the horizontal line at the top of the bar represents the standard deviation of ICAM-1, which is statistically significant. In FIG. 14, B, C, D, the horizontal axes indicate the groups, and the vertical axes indicate the mRNA levels of TNF-. alpha.IL-1. beta., and NLRP3, respectively.

As can be seen from FIG. 14, the mRNA levels of ICAM-1, TNF- α, IL-1 β, NLRP3 in the vascular tissues of the mice in the C57BL/6 mouse high-sugar and high-fat-fed group were significantly higher than those in the C57BL/6 mouse control group. However, the mRNA levels of ICAM-1, TNF- α, IL-1 β, NLRP3 in the vascular tissues of the C57BL/6 mice were significantly lower than those of the C57BL/6 mice high-sugar high-fat-fed group and the effect of adiponectin was associated with APPL1 when the mice were high-sugar high-fat-fed and administered to adiponectin-treated groups.

Respectively detecting the protein levels of a C57BL/6 mouse control group, a C57BL/6 mouse high-sugar high-fat feeding group, a C57BL/6 mouse high-sugar high-fat feeding group and adiponectin treatment group, an APPL 1-/-gene knockout mouse control group, an APPL 1-/-gene knockout mouse high-sugar feeding group, an APPL 1-/-mouse high-sugar high-fat feeding group and the ICAM-1 protein levels of the blood vessel tissues of 6 groups of model mice of the adiponectin treatment group by using 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 ICAM-1 (purchased from Abcam, usa); incubation secondary antibody (purchased from Abcam, usa); developing and using ChemiDoc^TMThe Touch imaging system (Bio-Rad) imaged, resulting in FIG. 15.

As shown in FIG. 15, Panel A is a western-blot of vascular tissue ICAM-1 of samples of C57BL/6 mouse control group, C57BL/6 mouse high sugar and high fat fed group, C57BL/6 mouse high sugar and high fat fed group and adiponectin-treated group, APPL 1-/-knockout mouse control group, APPL 1-/-knockout mouse high sugar fed group, APPL 1-/-knockout mouse high sugar and high fat fed group and adiponectin-treated group 6 groups, wherein GAPDH is glyceraldehyde-3-phosphate dehydrogenase (i.e., internal reference), and the black area indicates the content of vascular tissue ICAM-1 and glyceraldehyde-3-phosphate dehydrogenase, and the larger the black area, the more ICAM-1 is contained in the vascular tissue of the mouse, the larger the Dalton (KD) is in atomic mass units. Panel B is a C57BL/6 mouse control group, C57BL/6 mouse high sugar and high fat fed group, C57BL/6 mouse high sugar and high fat fed group and adiponectin treated group, apl 1-/-knockout mouse control group, apl 1-/-knockout mouse high sugar fed group, apl 1-/-knockout mouse high sugar fed group and protein level histogram of vascular tissue ICAM-1 of samples from 6 groups of adiponectin treated group, black area size was plotted using Image Lab Software (Bio-Rad), statistical analysis was performed using Graph Prism8.0 Software (Graph pad Software, San Diego, CA) and histogram was plotted. In the figure, the horizontal axis represents groups, the vertical axis represents the protein level of ICAM-1 (with glyceraldehyde-3-phosphate dehydrogenase as a reference), the horizontal line at the top of the bar represents the standard deviation of the ICAM-1 protein level, and the top horizontal line and asterisk in the figure represent the comparison between the two groups covered by the horizontal lines, which is statistically significant.

As can be seen from FIG. 15, the protein level of ICAM-1 in the vascular tissue of mice fed with high fat was significantly higher than that of ICAM-1 in the vascular tissue of normal control mice, while the protein level of ICAM-1 in adiponectin-perfused diabetic mice was inhibited, and this effect of adiponectin was dependent on APPL 1.

EXAMPLE VI human umbilical vein endothelial cell assay-mechanism of adiponectin to inhibit vascular inflammatory response

1：

Human umbilical vein endothelial cells were purchased from the national scientific and technological resource sharing service platform Beijing cooperative cell center, and the cells were expressed as 1x10⁵The density of/ml was seeded in six well plates. The human umbilical vein endothelial cells were divided into a blank control group, a 15-minute stimulation group, a 30-minute stimulation group, a 60-minute stimulation group, a 90-minute stimulation group, and a 120-minute stimulation group. Wherein, the human umbilical vein endothelial cells of the blank control group are treated by physiological saline, the human umbilical vein endothelial cells of the 15-minute stimulation group are cultured for 15 minutes by using adiponectin, the human umbilical vein endothelial cells of the 30-minute culture group are cultured for 30 minutes by using adiponectin, the human umbilical vein endothelial cells of the 60-minute stimulation group are cultured for 60 minutes by using adiponectin, and the human umbilical vein endothelial cells of the 90-minute culture group are cultured for 60 minutes by using adiponectinThe culture was carried out for 90 minutes, and the human umbilical vein endothelial cells in the 120-minute culture group were cultured with adiponectin for 120 minutes.

The cells were harvested and the Cell membrane, cytosolic and nuclear fractions were separated using a Cell fraction isolation Kit (available from QIAGEN, germany, Qproteome Cell components Kit) according to the Kit instructions, as follows:

(1) dissolving protease inhibitor solution, lysis solution, and extraction buffer solution;

(2) after dissolving, adding the protease inhibitor into the lysate and the buffer solution;

(3) transferring the cell suspension into a 15ml centrifuge tube, centrifuging for 10min at the temperature of 4 ℃ at 500g, and removing the supernatant;

(4) suspending the cell precipitate in 2ml of precooled PBS, mixing uniformly, centrifuging at 500g and 4 ℃ for 10min, and discarding the supernatant;

(5) repeating the step (4);

(6) suspending the cell pellet in 1ml of precooled lysate, and incubating for 10min at 4 ℃;

(7) centrifuging with a centrifuge at 1000g and 4 deg.C for 10 min;

(8) transferring the supernatant into a new centrifugal tube, and placing on ice, wherein the protein is cytoplasmic protein;

(9) suspending the cell pellet in 1ml of pre-cooled buffer CE2, and incubating at 4 ℃ for 30 min;

(10) centrifuging at 4 deg.C for 10min with a centrifugal force of 6000 g;

(11) transferring the supernatant into a new centrifugal tube, and placing on ice, wherein the protein is the cell membrane protein;

(12) adding 7 mul

Nuclean and 13. mu. lddH2O into the cell pellet, incubated for 15min at room temperature;

(13) adding 500 μ l of pre-cooled buffer solution, mixing well, and incubating at 4 deg.C for 10 min;

(14) centrifuging at 6800g for 10min at 4 deg.C;

(15) transferring the supernatant into a new centrifuge tube, and placing on ice, wherein the protein is the nucleoprotein;

detecting the protein distribution levels of APPL1 in a blank control group, a 15-minute stimulation group, a 30-minute stimulation group, a 60-minute stimulation group, a 90-minute stimulation group and a 120-minute stimulation group respectively 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 primary antibody is apl 1 (available from Cell Signaling Technology, usa); incubation secondary antibody (purchased from Abcam, usa); developing and using ChemiDoc^TMThe Touch imaging system (Bio-Rad) imaged, resulting in FIG. 16.

Fig. 16 is a western-blot graph of the protein distribution levels of the blank control group, 15-minute stimulation group, 30-minute stimulation group, 60-minute stimulation group, 90-minute stimulation group, and 120-minute stimulation group APPL1, the black area indicating the cell APPL1 content, and the larger the black area, the more the cell component APPL1 content, the larger the dalton (KD) is in atomic mass units. In FIG. 16, the first Western blot band is a distribution diagram of APPL1 in the cell membrane, the second Western blot band is a distribution diagram of APPL1 in the cytoplasm, and the third Western blot band is a distribution diagram of APPL1 in the nucleus.

As can be seen in figure 16, adiponectin promoted the engraftment of APPL1 in a time-dependent manner.

Carrying out laser confocal scanning on human umbilical vein endothelial cells of a blank control group, a 60-minute stimulation group and a 120-minute stimulation group respectively, observing the distribution condition of APPL1 in cells, namely completely sucking a cell culture medium, and adding 300 mu l of PBS into each hole for cleaning; fixing with 4% paraformaldehyde at room temperature for 20 min; rinsing with PBS at room temperature for three times, each time for 5 min; blocking with 5% BSA at room temperature for 20 min; removing the blocking solution, adding an anti-APPL 1 antibody, and incubating overnight at 4 ℃; rinsing with PBS at room temperature for 5min for three times; adding Cy5 labeled secondary antibody, and incubating for 1h at 37 ℃ in the dark; rinsing with PBS at room temperature for three times, each time for 5 min; adding DARQT5 staining solution, and reacting at room temperature for 5 min; rinsing with PBS at room temperature for three times, each time for 5 min; the fluorescence quenching blocking agent was blocked, and the image was observed and collected under a confocal laser microscope to obtain fig. 17.

In FIG. 17, the first row shows the confocal laser staining of the cells in the blank control group, the second row shows the confocal laser staining of the cells in the 60 minute stimulation group, and the third row shows the confocal laser staining of the cells in the 120 minute stimulation group, wherein the gray dots in the figure indicate the APPL1 protein, and the larger circular spots indicate the nuclear parts. As can be seen in fig. 17, adiponectin promoted the engraftment of APPL1 in a time-dependent manner.

The RNA-seq technology analysis is adopted to detect that the adiponectin activates related transcription factors in an APPL1 dependent mode, wherein the detection step of the RNA-seq technology comprises the following steps: extracting RNA of the vascular tissue; PCR amplification; sequencing; controlling the quality; data and processing gave figure 18.

As shown in FIG. 18, the graphs-1 to 1 represent, from bottom to top, differential gene expression down-and up-regulation. From left to right, each three boxes represent a group of samples, which are the differential expression of the vascular tissue transcription factor-related gene of the normal control group, the differential expression of the vascular tissue transcription factor-related gene of the high-glucose and high-lipid mouse, the differential expression of the vascular tissue transcription factor-related gene of the adiponectin-treated high-glucose and high-lipid mouse, the APPL 1-/-gene-knockout mouse is fed with high-glucose and high-lipid and the differential expression of the vascular tissue transcription factor-related gene is given to adiponectin treatment.

As can be seen in FIG. 18, high-sugar and high-fat inhibited the partial transcription factor enrichment, while adiponectin promoted the transcription factor TCF/LEF enrichment, a process related to APPL 1.

2：

The human umbilical vein endothelial cells are inoculated on a six-well plate at the density of 1x105/ml, and are divided into a blank control group, a high-fat high-sugar medium stimulation group, a high-fat high-sugar medium + adiponectin stimulation group, and knockdown siAPPL1 (purchased from Jima gene, Suzhou Co., Ltd.) + the high-fat high-sugar medium + adiponectin stimulation group, wherein the high-fat high-sugar medium is stimulated for 72 hours, and the adiponectin is stimulated for 24 hours. The blank control group used a common medium whose composition comprised endothelial cell culture medium (purchased from ScienCell corporation, USA) + 10% fetal bovine serum (purchased from Life technologies, Germany) + 1% antibiotic Penicillin-Streptomyces (purchased from Thermo Fisher Scientific, USA); the high-sugar, high-fat medium components contained 4.5g/L D-glucose medium (available from Life technologies, Germany) + 5% high-fat working solution (0.01397g sodium palmitate and 1g bovine serum albumin in 10ml phosphate buffered saline PBS as working solution) + 10% fetal bovine serum (available from Life technologies, Germany) + 1% antibiotic Penicillin-Streptomyces (available from Thermo Fisher Scientific, USA).

Transcription factor activation assay plate array II (purchased from signalis, Sunnyvale, CA) was used to assay transcription factor activity in HUVEC cells. As shown in FIG. 19, the diagram from bottom to top (-2) indicates differential gene expression down-regulation and up-regulation. From left to right, each three boxes represent a group of samples, which are respectively differential expression of a gene related to a blank control group transcription factor, differential expression of a gene related to a high-fat high-sugar culture medium stimulation group transcription factor, differential expression of a gene related to a high-sugar high-fat culture medium plus adiponectin stimulation group transcription factor, knocking down APPL1 plus high-sugar high-fat culture medium and giving differential expression of a gene related to adiponectin treatment vascular tissue transcription factor.

As can be seen in FIG. 19, high lipids inhibit partial transcription factor activation, while adiponectin promotes transcription factor activation, a process related to APPL 1.

In agreement with the grouping of human umbilical vein endothelial cells in FIG. 19, mRNA levels of transcription factors in HUVEC cells were analyzed using human transcription factors (to be purchased from Qiagen, USA). The detailed steps are explained in the term "human transcription factor detection kit".

As shown in FIG. 20, panel A is a scattergram of a human umbilical vein endothelial cell blank control group and a high-fat high-sugar medium-stimulated group, wherein a point located above three straight lines (left side of the straight line) indicates the mRNA level of a high-fat high-sugar up-regulated transcription factor compared to a normal control group; black dots on the straight line indicate no difference in the transcription factor mRNA levels between the normal control group and the high-fat high-sugar stimulated group; the point below the line (right side of line) indicates the mRNA level of the high-sugar high-fat downregulated transcription factor compared to the normal control group, and the notation TCF7L2 indicates the mRNA level that is consistent with high-sugar repression. B is a scatter diagram of a human umbilical vein endothelial cell high-fat and high-sugar medium stimulation group, a high-fat and high-sugar medium + adiponectin stimulation group, wherein a point (left side of a straight line) positioned above three straight lines represents the mRNA level of an adiponectin up-regulated transcription factor compared with the high-sugar and high-fat medium stimulation group; the black dots on the straight line indicate no difference in the high fat and high sugar plus adiponectin stimulated group transcription factor mRNA levels; the point below the line (right side of line) indicates that adiponectin down-regulates the mRNA level of the transcription factor compared to the high-fat high-sugar stimulated group, and the notation TCF7L2 indicates the mRNA level consistent with adiponectin promoting it. Panel C is a scatter plot of human umbilical vein endothelial cell high-fat high-sugar medium + adiponectin stimulated group, knocked-down APPL1+ high-fat high-sugar medium + adiponectin stimulated group, where the point located above the three straight lines (left side of the straight line) indicates that the knock-down APPL1+ high-fat high-sugar medium + adiponectin stimulated group up-regulates the mRNA level of transcription factor compared to the high-sugar high-fat medium + adiponectin stimulated group; black dots on the straight line indicate no difference in the transcription factor mRNA levels of the high-fat high-sugar medium + adiponectin stimulated group and the knockdown apl 1+ high-fat high-sugar medium + adiponectin stimulated group; the point below the line (right side of the line) indicates that knock-down APPL1+ high-fat high-sugar medium + adiponectin stimulated group down-regulates the mRNA level of the transcription factor compared to high-fat high-sugar medium + adiponectin stimulated group, and the notation TCF7L2 indicates that it is consistent with adiponectin promoting its mRNA level.

3：

The human umbilical vein endothelial cells are inoculated on a six-well plate at the density of 1x105/ml, and are divided into a blank control group, a high-fat high-sugar medium stimulation group, a high-fat high-sugar medium + adiponectin stimulation group, a knockdown siAPPL1 (from Suzhou Jima gene Co., Ltd.) + high-fat high-sugar medium + adiponectin stimulation group and a knockdown siAPPL1 (from Suzhou Jima gene Co., Ltd.) + high-fat high-sugar medium, wherein the high-fat high-sugar medium is stimulated for 72 hours, and adiponectin is stimulated for 24 hours.

Collecting cells, and respectively detecting the protein distribution level of CD44 in a blank control group, a high-fat high-sugar medium stimulation group, a high-fat high-sugar medium + adiponectin stimulation group, a knockdown siAPPL1+ high-fat high-sugar medium + adiponectin stimulation group and a knockdown siAPPL1+ high-fat high-sugar medium culture medium by using 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 apl 1 (purchased from Cell Signaling Technology, usa); incubation twoAnti (available from Abcam, USA); developing and using ChemiDoc^TMThe Touch imaging system (Bio-Rad) imaged to obtain FIG. 21.

As shown in fig. 21, panel a is a western-blot diagram of protein distribution levels of the blank control group, the high-fat high-sugar medium stimulation group, the high-fat high-sugar medium + adiponectin stimulation group, the knock-down siAPPL1+ high-fat high-sugar medium + adiponectin stimulation group, and the knock-down siAPPL1+ high-fat high-sugar medium, where GAPDH is glyceraldehyde-3-phosphate dehydrogenase (i.e., internal reference), and black area indicates the content of cell CD44 and glyceraldehyde-3-phosphate dehydrogenase, and the larger black area indicates the greater content of cell component CD44, and dalton (KD) is atomic mass unit. Panel B is a bar Graph comparing the protein levels of the blank control group, the high-fat high-sugar medium stimulated group, the high-fat high-sugar medium + adiponectin stimulated group, the knockdown siAPPL1+ the high-fat high-sugar medium + adiponectin stimulated group, the knockdown siAPPL1+ the high-fat high-sugar medium cells CD44, the black area size using Image Lab Software (Bio-Rad), the statistical analysis using Graph Prism8.0 Software (Graph pad Software, San Diego, CA), and the bar Graph, wherein the horizontal axis represents the group category, the vertical axis represents the protein level of CD44 (with glyceraldehyde-3-phosphate dehydrogenase as a reference), the horizontal line at the top of the bar represents the standard deviation of the protein level of CD44, and the top horizontal line and asterisk in the figure represent the comparison of the two groups covered by the horizontal line, which has statistical significance.

As can be seen from figure 21, adiponectin promoted expression of CD44 protein in an apl 1-dependent manner.

After endothelial cells were cultured, RNA was extracted, and Real-time PCR was used to detect mRNA levels of inflammatory factors in the blank control group, the high-fat high-sugar medium-stimulated group, the high-fat high-sugar medium + adiponectin-stimulated group, and the knockdown siCD44+ high-fat high-sugar medium + adiponectin-stimulated group, respectively, to obtain fig. 22.

As shown in fig. 22, a graph a is a bar graph comparing the mRNA levels of TNF- α in the blank control group, the high-fat high-sugar medium stimulation group, the high-fat high-sugar medium + adiponectin stimulation group, and the knockdown siCD44 high-fat high-sugar medium + adiponectin stimulation group, wherein x represents that the mRNA level of TNF- α in the blank control group is 1 and the mRNA level of TNF- α in the two groups is different compared with the blank control group. # indicates that the mRNA level of TNF- α in the high-fat glucose medium-stimulated group was 1 and the mRNA level of TNF- α was different between the two groups, compared to the high-fat glucose medium-stimulated group. And & indicates that the mRNA level of TNF-alpha in the high-fat high-sugar medium + adiponectin stimulated group is 1 and the mRNA level of TMF-alpha is different between the two groups compared with the high-fat high-sugar medium + adiponectin stimulated group in the knockdown siCD44 high-fat high-sugar medium + adiponectin stimulated group. The horizontal axis represents the group, the vertical axis represents the mRNA level of TNF-. alpha.and the horizontal line at the top of the bar represents the standard deviation of TNF-. alpha.with statistical significance. Panel B, C and D in FIG. 22 correspond to Panel A, with the horizontal axes representing the groups and the vertical axes representing the mRNA levels of IL-1. beta., ICAM-1 and NLRP3, respectively.

As can be seen in fig. 22, the effect of adiponectin in inhibiting the vascular inflammatory response is associated with CD 44.

4：

The group model is established by adopting the method: FIG. 23 is a graph showing the levels of protein in CD44 in 6 groups of mice in the control group of C57BL/6 mice, the group of C57BL/6 mice fed high sugar and high fat, the group of C57BL/6 mice fed high sugar and high fat and adiponectin-treated mice, the control group of APPL 1-/-knockout mice, the group of APPL 1-/-knockout mice fed high sugar and high fat, and the group of APPL 1-/-mice fed high sugar and adiponectin-treated 6 groups of 6 models, respectively, by Western Blot technique.

As shown in fig. 23, the a-map is a western-blot of 6 sets of sample vascular tissue CD 44. Panel B is a bar Graph of protein levels of vascular tissue CD44 from group 6 samples, plotted as black areas using Image Lab Software (Bio-Rad), statistically analyzed using Graph Prism8.0 Software (GraphPad Software, San Diego, Calif.) and plotted as a bar Graph. The horizontal axis represents groups, the vertical axis represents the protein level of CD44 (with glyceraldehyde-3-phosphate dehydrogenase as a reference), the horizontal line at the top of the bar represents the standard deviation of the CD44 protein level, and the top horizontal line and asterisk in the figure represent the comparison between the two groups covered by the horizontal lines, which is statistically significant.

As can be seen from fig. 23, adiponectin in mouse vascular tissue promoted expression of CD44 protein in an apll 1-dependent manner.

Example seven: test for comparing adiponectin and CD44 levels in patient serum

Patients with initial diagnosis of diabetes mellitus are continuously collected at an Anzhen hospital in Beijing, affiliated to the university of capital medical science, according to the Chinese diabetes diagnosis standard: firstly, fasting blood glucose (FPG) is more than or equal to 7.0 mmol; ② for those with typical diabetes symptoms (polyuria, polydipsia and unexplained weight loss), the arbitrary blood sugar is more than or equal to 11.1 mmol/L. Diabetes can be diagnosed by meeting one of the above criteria. The exclusion criteria are secondary hypertension such as kidney disease, pheochromocytoma, primary aldosteronism and the like; ② severe arrhythmia; moderate and severe cardiac insufficiency; fourthly, active liver dysfunction or ALT or AST more than 3 times of normal upper limit is found by physical examination; malignant diseases: a tumor; sixthly, anemia is generated; acute and chronic inflammatory diseases: including rheumatic arthritis, rheumatoid arthritis, etc. The remaining 74 of these diabetic patients were used as the observation group.

30 subjects were randomly selected as normal control groups from the population who had been subjected to health examination in the same hospital examination center and had normal blood sugar. Collecting multiple routine test indexes such as blood lipid, blood glucose and blood, and carefully inquiring the disease history such as cardiovascular diseases, liver and kidney diseases and family history.

After 8-10 hours fasting, 10ml blood samples were taken intravenously, centrifuged at 3000rpm for 10 minutes, and serum was extracted and frozen in a-80 freezer for all patients.

The adiponectin levels in the sera of the subjects in the observation group and the normal control group were determined by a high-sensitivity enzyme-linked immunosorbent assay (ELISA) (kit from Cat #8B5D6EB29B, Cloud-Clone Corp), respectively. The adiponectin in the ELISA kit is coated in a 96-hole microporous plate in advance to form a solid phase carrier. Adding a standard substance and a sample into the micropore, wherein the adiponectin is combined with an antibody coated on a solid phase carrier, then adding a biotinylated adiponectin antibody, washing the unbound biotinylated antibody, adding horseradish peroxidase (HRP) labeled avidin, washing again, and adding a3, 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 the adiponectin concentration in the sample. Absorbance (o.d. value) was measured at a wavelength of 450nm with a microplate reader to obtain a standard curve, and then the adiponectin concentration in the sample was calculated from the standard curve and statistically analyzed with SPSS software (IBM corp., Armonk, NY, USA), giving fig. 24.

FIG. 24 is a bar graph comparing the serum adiponectin levels in the subjects observed in the groups and in the normal control group, wherein the horizontal axis represents the group, the vertical axis represents the adiponectin level (ng/ml), the horizontal line above the bar represents the standard deviation of the adiponectin level, and the asterisks indicate that the experiment and the experimental results are statistically significant. It can be seen that the content of adiponectin in the serum of the observation group subject is significantly lower than that of the normal control group subject, so that the expression level of adiponectin in the serum of the diabetic patient is significantly reduced, and adiponectin is inhibited in the diabetic patient.

FIG. 25 is a bar graph comparing the levels of CD44 in the sera of subjects in the observation group and normal control group, wherein the horizontal axis represents the group, the vertical axis represents the level of CD44 (ng/ml), the horizontal line above the bar represents the standard deviation of the level of CD44, and the asterisks indicate that the experiment and the results of the experiment are statistically significant. As can be seen, the content of CD44 in the serum of the observed group patients is obviously lower than that of CD44 in the serum of the normal control group subjects, so that the expression level of CD44 in the serum of the diabetic patients is remarkably reduced, and CD44 is inhibited in the diabetic patients.

Fig. 26 is a graph showing correlation analysis between CD44 and adiponectin in the serum of diabetic patients, in which the horizontal axis represents the level of CD44 in the serum and the vertical axis represents the content of adiponectin in the serum, and SPSS software was used to determine the correlation coefficient r of 0.259 and p of 0.0247, and when p is less than 0.05, there was a correlation between the two. It can be seen that there is a correlation between serum adiponectin levels and CD44 levels in diabetic patients.

The foregoing description of the embodiments of the present application has been presented for purposes of illustration and description and is not intended to be exhaustive or limited to the embodiments disclosed. Many modifications and variations will be apparent to those of ordinary skill in the art without departing from the scope and spirit of the described embodiments. The terminology used herein is chosen in order to best explain the principles of the embodiments, the practical application, or improvements made to the technology in the marketplace, or to enable others of ordinary skill in the art to understand the embodiments disclosed herein. The scope of the application is defined by the appended claims.

Claims (7)

1. Application of a substance for promoting CD44 level in preparing a product for treating vascular dermatitis caused by hypertension or hyperlipidemia by promoting CD 44.

2. The use according to claim 1, wherein the substance that promotes CD44 levels is used in combination with other drugs for the treatment of vascular endothelial inflammation in the product.

3. The use of claim 1, wherein the agent that promotes CD44 levels is a CD44 promoter.

4. The use of claim 3, wherein the substance that promotes the level of CD44 is a CD44 recombinant protein.

5. The use of claim 1, wherein the substance promoting the level of CD44 promotes the expression of downstream target gene CD44 in a manner of promoting APPL1 to enter the nucleus and activating transcription factor TCF/LEF.

6. The use according to claim 1, wherein the product for the treatment of vascular endothelial inflammation further comprises an adjuvant.

7. The use according to claim 1, wherein the product for treating vascular endothelial inflammation is an injection preparation, an oral tablet or an oral capsule.

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