CN115779087A

CN115779087A - Application of GHRH antagonist in preparation of medicine for preventing and treating vascular diseases

Info

Publication number: CN115779087A
Application number: CN202211385996.3A
Authority: CN
Inventors: 余红; 张弛
Original assignee: Binjiang Research Institute Of Zhejiang University; Zhejiang University ZJU
Current assignee: Binjiang Research Institute Of Zhejiang University; Zhejiang University ZJU
Priority date: 2022-11-07
Filing date: 2022-11-07
Publication date: 2023-03-14

Abstract

The invention relates to the field of biomedicine, and discloses application of a GHRH antagonist in preparing a medicament for preventing and treating vasculopathy. The GHRH antagonist is found for the first time to increase vascular endothelial tight junction protein, improve the level of vascular endothelial oxidative stress, prevent and reverse vascular endothelial injury, provide a new strategy for preventing and treating vascular lesions and have important clinical treatment significance; in addition, the GHRH antagonist can prevent and treat the weight, the motor ability and the cardiac function reduction caused by hyperglycemia, and is expected to become a new prevention and treatment means for hyperglycemia complications.

Description

Application of GHRH antagonist in preparation of medicine for preventing and treating vasculopathy

Technical Field

The invention relates to the field of biomedicine, in particular to application of a Growth Hormone Releasing Hormone (GHRH) antagonist in preparing a medicament for preventing and treating vasculopathy.

Background

The vascular endothelium is a continuous monolayer of cells formed by different types of adherent structures or cell-to-cell connections and plays an important role in driving angiogenesis, controlling the exchange of substances between blood and tissue, regulating vascular tone, regulating inflammatory reactions in vivo, as well as anticoagulation and antithrombotic formation. However, the continuous large amount of stimulation easily causes damage to the vascular endothelium, so that the oxidative stress of the vascular endothelium generates a large amount of ROS, the function of the vascular endothelium is influenced, and the vascular diseases are caused. For example, studies have shown that hyperglycemia directly or indirectly impairs normal vascular endothelial function, initiates or aggravates vascular lesions, and has "hyperglycemic memory", chronic hyperglycemia in the long term leads to changes in vascular structure and function, and short-term hyperglycemia in the long term has a long-term effect on blood vessels. The search for the medicine for preventing and reversing vascular endothelial injury provides a new strategy for preventing and treating vascular diseases, and has important significance.

Growth Hormone Releasing Hormone (GHRH) is a neuroendocrine peptide composed of 44 amino acids, which is mainly secreted from hypothalamus, and its receptor (GHRH-R) is widely present in tissues and organs such as pituitary gland, lung, eye, placenta, etc. The natural GHRH has a very short half-life in vivo, and schallely, a co-worker of the present inventors, professor of miami university of the united states, invented GHRH analogs of 29 amino acids as growth hormone releasing hormone agonists and antagonists. Among them, GHRH antagonists can competitively bind to GHRH-R, and it has been found that they have effects of inhibiting tumor cell division, reducing intraocular inflammatory reaction caused by endotoxin, improving decrease of brain oxygen supply during aging, etc., but they have not been reported to play a role in preventing and treating vascular diseases.

Disclosure of Invention

In order to solve the technical problems, the invention provides an application of a GHRH antagonist in preparing a medicament for preventing and treating vasculopathy. The GHRH antagonist can increase vascular endothelial tight junction protein, improve the level of vascular endothelial oxidative stress and prevent and reverse vascular endothelial injury, thereby providing a new strategy for preventing and treating vascular lesions and having important clinical treatment significance.

The invention also provides application of the GHRH antagonist in preparing a medicament for preventing and treating organ damage caused by hyperglycemia. The GHRH antagonist can prevent organ damage caused by hyperglycemia, further improve symptoms such as weight loss, exercise capacity reduction, cardiac contractility reduction and the like, and is expected to become a new treatment means for hyperglycemia complications.

The specific technical scheme of the invention is as follows:

the invention provides an application of a growth hormone releasing hormone antagonist in preparing a medicament for preventing or treating vasculopathy.

Preferably, the vascular disorder comprises vascular endothelial injury.

Preferably, the vascular disorder comprises a decrease in vascular endothelial claudin.

Preferably, the vascular disorder comprises vascular endothelial cell oxidative stress.

The research of the invention group finds that the Growth Hormone Releasing Hormone (GHRH) antagonist has the prevention and treatment effect on vascular lesions, can improve the oxidative stress level of vascular endothelial cells, reduce Reactive Oxygen Species (ROS) in the vascular endothelial cells, and simultaneously can increase vascular endothelial tight junction protein, prevent and reverse vascular endothelial injury, improve the function of the vascular endothelium and reduce the permeability of the vascular endothelium. Preferably, the vascular disorder is a vascular disorder caused by hyperglycemia.

Preferably, the medicament comprises the following (a) and/or (b):

(a) A growth hormone releasing hormone antagonist;

(b) Pharmaceutically acceptable salts and/or esters of a growth hormone releasing hormone antagonist.

Further, the medicament further comprises one or more of pharmaceutically acceptable carriers, excipients, solvents and buffers.

Preferably, the dosage form of the drug includes, but is not limited to, injection, oral preparation, smearing preparation, inhalation preparation, implant micro-pump, eye drop, lyophilized powder injection, tablet, pill, granule, hard capsule, syrup, soft capsule, ointment, cream, microneedle, patch or spray.

Preferably, the drug is administered by a method including, but not limited to, subcutaneous injection, intravenous injection, intramuscular injection, micropump implantation, oral nasal pulmonary inhalation, eye drip irrigation, painting, skin patch, swallowing, or topical application.

Preferably, the ghrelin antagonist is a MIA series, AVR series, MZ series, or JV series GHRH antagonist, but is not limited to the above series.

Preferably, the object of application of the medicament is human or animal.

Furthermore, the human body usage amount of the medicine is 0.01 mu g/kg-1mg/k each time. Further, the frequency of use may be 1 or more times per day.

In a second aspect, the present invention provides the use of a growth hormone antagonist in the manufacture of a medicament for the prevention or treatment of body or organ damage caused by hyperglycemia.

Hyperglycemia causes damage to the body and organs, and in turn causes weight loss, decreased levels of exercise and endurance, and decreased systolic function. However, the GHRH antagonist can relieve the damage of organisms and organs caused by hyperglycemia, improve the symptoms to a certain extent and maintain normal body weight, exercise capacity and cardiac function.

Preferably, the injury to the body or organ is caused by a vascular disorder.

The GHRH antagonist can prevent and treat vascular diseases in target organs and the whole body, protect the vascular barrier function and the number of blood vessels in the target organs and the whole body and increase the blood oxygen supply. Thus, the protective effects of GHRH antagonists on target organs and organisms can be functionally protective by acting on blood vessels, but not limited to improving target organ and organism function by acting on blood vessels.

Preferably, the organ comprises a heart, brain, lung or peripheral extremity.

Compared with the prior art, the invention has the following advantages:

(1) The GHRH antagonist is found for the first time to be capable of inhibiting the vascular endothelial oxidative stress, increasing the level of vascular endothelial tight junction protein, preventing and reversing vascular endothelial injury and having important significance for preventing and treating vascular lesions;

(2) The GHRH antagonist can prevent and treat the damage of organisms and organs caused by hyperglycemia, improve the symptoms of weight, exercise capacity, cardiac function reduction and the like caused by the damage of the organisms or the organs, and is expected to become a new prevention and treatment means for hyperglycemia complications.

(3) The GHRH antagonist has the characteristics of good stability and high drug activity in the drugs for preventing and treating the vasculopathy; meanwhile, the medicine has the advantages of simple preparation, obvious effect and small side effect.

Drawings

Figure 1 is a graph of experimental results relating to the effect of GHRH antagonists on vascular endothelial injury. Wherein: FIG. 1A is a schematic diagram of the experiment of the systemic vascular permeability of hyperglycemic mice modeled by FITC-Dextran detection of streptozotocin STZ; FIG. 1B is a FITC-Dextran resident fluorescence quantitative histogram of different organ tissues of different treatment groups; FIG. 1C is a representative image of Dextran (green) and CD31 (red) immunofluorescent staining of cardiac tissue of different treatment groups; FIG. 1D is a representative image of Dextran (green) and CD31 (red) immunofluorescent staining of lung tissue for different treatment groups; FIG. 1E is representative images of Dextran (green) and CD31 (red) immunofluorescent staining of brain tissue of different treatment groups; FIG. 1F shows the expression level of Claudin (CLDN 5, ZO-1,occludin) in different organ tissues of different treatment groups.

FIG. 2 is the results of experiments relating to the effect of GHRH antagonists on mouse body weight, cardiac contractile function and motor ability. Wherein, fig. 2A is a graph of weight change at different time points for different treatment groups; FIG. 2B is a statistical graph of systolic function analysis for different treatment groups two months after cardiac ultrasound assessment administration; FIG. 2C is a representative image of the assessment of exercise capacity on a treadmill for different treatment groups after two months of dosing; fig. 2D is a statistical graph of the motion duration for different processing groups.

FIG. 3 is a graph of experimental results relating to the effect of GHRH antagonists on blood glucose and urine glucose levels in hyperglycemic mice. Wherein, fig. 3A is the blood glucose level change at different time points for different treatment groups; figure 3B is the urine glucose levels of the different treatment groups after two months of administration.

FIG. 4 is a graph of experimental results relating to the effect of GHRH antagonists on vascular endothelial permeability, cellular oxidative stress levels, and tight junction protein. Wherein, FIG. 4A is a statistical graph of the flow detection of DCFH-A fluorescent probe-labeled ROS in HUVEC cells of different treatment groups; FIG. 4B is a schematic of the experiment with FITC-dextran penetrating the HUVEC cell layer (left) and the results of the assay (right); FIG. 4C is a representative image of immunofluorescence staining for Claudin CLDN5 (green) and ZO-1 (red) in HUVEC cells from different treatment groups; FIG. 4D shows the expression level of the tight junction protein of HUVEC cells in different treatment groups measured by Western Blot.

Figure 5 is the results of experiments relating to the effect and mechanism of GHRH antagonists on GHRHR expression levels. Wherein, FIG. 5A is a Western Blot for detecting GHRHR protein expression quantity in HUVEC cells of different treatment groups; FIG. 5B shows the level of PKC/P65 phosphorylation in HUVEC cells from different treatment groups as measured by Western Blot; FIG. 5C is a Western Blot to detect the change of GHRHR expression level in NF-. Kappa.B-siRNA knockdown HUVEC cells; FIG. 5D is a dual luciferase reporter assay validating NF-. Kappa.B as a transcription factor for GHRHR.

FIG. 6 is the results of experiments relating to the effect of GHRH antagonists on cAMP/PKC/CREB pathway phosphorylation levels and matrix metalloproteinase expression levels. Wherein, figure 6A is a different processing group transcriptomics sequencing differential gene KEGG pathway enrichment analysis; FIG. 6B is a Western Blot assay for PKC/CREB phosphorylation levels in HUVEC cells of different treatment groups; FIG. 6C shows the detection of MMP enzyme activity in HUVEC cells of different treatment groups by gelatinase spectrometry; FIG. 6D shows the difference in the expression level of MMP and tight junction-associated proteins in HUVEC cells of different treatment groups measured by Western Blot.

Detailed Description

The present invention will be further described with reference to the following examples.

General examples

Use of a ghrelin antagonist in the manufacture of a medicament for the prevention or treatment of vasculopathy.

Optionally, the vascular disorder comprises one or more of vascular endothelial injury, a decrease in vascular endothelial claudin, oxidative stress of vascular endothelial cells.

Optionally, the vascular disorder is a vascular disorder caused by hyperglycemia.

Optionally, the medicament comprises the following (a) and/or (b):

(a) A growth hormone releasing hormone antagonist;

Optionally, the medicament comprises one or more of a pharmaceutically acceptable carrier, excipient, solvent and buffer in addition to (a) and/or (b) above.

Optionally, the medicament is in the form of injection, oral preparation, liniment preparation, inhalant preparation, implanted micro-pump, eye drop, lyophilized powder injection, tablet, pill, granule, hard capsule, syrup, soft capsule, ointment, cream, micro-needle, patch or spray, and is administered by subcutaneous injection, intravenous injection, intramuscular injection, micro-pump implantation, oro-nasal-pulmonary inhalation, eye drip irrigation, liniment, skin patch, swallowing or topical application.

Optionally, the growth hormone releasing hormone antagonist is a MIA series, AVR series, MZ series, or JV series GHRH antagonist.

Optionally, the subject of application of the medicament is a human or an animal.

Optionally, the amount of the drug applied to human body is 0.01 μ g/kg-1mg/k per time. Further, the frequency of use may be 1 or more times per day.

Use of a growth hormone antagonist for the manufacture of a medicament for the prevention or treatment of organ damage or weight loss or reduced exercise capacity or reduced cardiac function.

Optionally, the organ damage or weight loss or reduced exercise capacity or reduced cardiac function is caused by vasculopathy.

Optionally, the organ comprises a heart, brain, lung, or peripheral limb.

DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION

The present invention is illustrated below by means of specific examples, which use GHRH antagonists in the MIA602 series, but the scope of protection of the present invention is not limited thereto; in the present embodiment, the STZ model is used to model hyperglycemia mice, but the scope of the present invention is not limited thereto; the evaluation of the protection of the body and target organs by the drug application in this example relates to the functions of the heart, brain, kidney, lung, movement and body weight, but the scope of the invention is not limited thereto.

The MIA602 amino acid sequence is:

PhAcAdaTyr-DArg-Asp-Ala-Ile-Phe(F)5-Thr-Ala-Har-Tyr(Me)-His-Orn-Val-Leu-Abu-Gln-Leu-Ser-Ala-His-Orn-Leu-Leu-Gln-Asp-Ile-Nle-DArg-Har-NH ₂

(i.e., phAcAda-tyrosine-D-arginine-aspartic acid-alanine-isoleucine-phenylalanine-threonine-alanine-homoarginine-tyrosine-histidine-ornithine-valine-leucine-2-aminobutyric acid-glutamine-leucine-serine-alanine-histidine-ornithine-leucine-glutamine-aspartic acid-isoleucine-norleucine-D-arginine-homoarginine-NH ₂ ). Wherein PhAcAdaTyr represents that the N end of tyrosine is modified by PhAcAda, phe (F) 5 represents the benzene ring perfluorinated substitution of a phenylalanine side chain, tyr (Me) represents the phenolic hydroxyl methylation of a tyrosine side chain, and Har-NH ₂ Denotes the carboxy group of homoarginine-CONH ₂ And (c) substitution, the structural formula of the residue is as follows:

example 1: effect of GHRH antagonists on vascular endothelial injury

And carrying out small-dose intraperitoneal injection on the mice for multiple times until the blood sugar is increased to 17.1mmol/L, and completing the construction of a hyperglycemia model. Normal and hyperglycemic mice were dosed with 5 μ g per day of MIA602 (designated "STZ + MIA 602") or Vehicle control (designated "STZ + Vehicle") per mouse by subcutaneous injection, and 5 μ g per day of Vehicle (designated "CTRL" group) per mouse by subcutaneous injection. Two months after administration, mice were examined for systemic vascular permeability by tail vein injection using FITC-labeled Dextran of 70kDa size as follows:

respectively extracting different organ tissues for grinding, detecting FITC-Dextran resident fluorescence values (shown in figure 1A) of different organ tissues of different treatment groups by using a multifunctional microplate reader to extract Dextran fluorescence retained in the tissues, and obtaining a detection result shown in figure 1B. As can be seen from fig. 1B: compared with normal mice (CTRL group), fluorescence quantity of organs rich in blood vessels such as kidney, heart, lung, brain and the like of the modeling groups (STZ + vehicle group and STZ + MIA602 group) is obviously increased; after the MIA602 treatment, the STZ + MIA602 group had significantly reduced penetration compared to the STZ + vehicle group except that the kidney was not significantly changed. This indicates that: the MIA602 can obviously improve the vascular permeability of part of organs of STZ molding hyperglycemic mice.

Mouse heart tissue was sectioned and then immunofluorescent stained, and the results are shown in FIG. 1C. As can be seen from fig. 1C: the Dextran labeled with green fluorescence of the model group was significantly increased and the expression of the vascular endothelial marker CD31 labeled with red fluorescence was decreased compared to normal mice; compared with the STZ + vehicle group, the Dextran of the green fluorescence mark of the STZ + MIA602 group is obviously reduced, and at the same time, the expression level of the CD31 of the red fluorescence mark is increased (the numbers of the red fluorescence and the green fluorescence are difficult to accurately reflect due to the failure of color in the drawing of the specification, and can be obviously seen in the color original image). This indicates that: hyperglycemia causes increased permeability of cardiac vessels, and the number and function of blood vessels are destroyed; the MIA602 can protect the vascular barrier function and the number of blood vessels in the cardiovascular disease caused by hyperglycemia, increase the myocardial blood supply and oxygen supply, enhance the capabilities of cardiac contraction, relaxation, blood pumping and the like, and improve the functions of the heart in various aspects.

The lung tissue of the mice was sectioned and immunofluorescent stained, and the results are shown in FIG. 1D. As can be seen from fig. 1D: the green fluorescence labeled Dextran of the STZ + vehicle group is obviously increased, while the expression of the red fluorescence labeled endothelial marker CD31 is obviously reduced, which shows that the blood vessel density of the lung is reduced after the model is made, the blood vessel permeability is increased, and the blood vessel function is destroyed; the blood vessel density and the blood vessel permeability of the STZ + MIA602 group are improved to a certain extent compared with each other (the quantity of red fluorescence and green fluorescence is difficult to accurately reflect due to the fact that the blood vessel density and the blood vessel permeability cannot be colored in the drawing of the specification, and the quantity can be obviously seen in a colored original drawing). This indicates that: hyperglycemia also disrupts the barrier function of pulmonary vessels, and MIA602 can protect against pulmonary vascular lesions caused by hyperglycemia. With the maintenance of the blood vessel barrier function, alveolar cells, interstitial cells and the like obtain sufficient blood oxygen supply, and the lung is enhanced to play the functions of breathing and gas exchange.

Mouse brain tissue was sectioned and then immunofluorescent stained, and the results are shown in FIG. 1E. As shown in fig. 1E: compared with the normal group of mice, the green fluorescence labeled Dextran of the STZ + vehicle group is obviously increased, and the expression of the red fluorescence labeled endothelial marker CD31 is obviously reduced, which shows that the number of the blood vessels of the brain after modeling is also obviously reduced, the permeability of the blood vessels is increased, and the functions of the blood vessels are damaged; the blood vessel density and the blood vessel permeability of the mice treated by the MIA602 are improved to a certain extent (the quantity of red fluorescence and green fluorescence is difficult to accurately reflect in the attached drawing of the specification due to the fact that the mice cannot have colors, and the quantity can be obviously seen in a colored original drawing). This indicates that: hyperglycemia also destroys the vascular barrier of brain tissue, and MIA602 treatment can reverse the hyperglycemia-induced cerebrovascular damage. When the blood vessel barrier function is kept stable, brain cells obtain sufficient blood oxygen supply, so that the incidence rate of cerebral apoplexy, cerebral infarction and the like can be reduced, and the functions of all aspects of the brain can be enhanced.

Tissue proteins from different organs were extracted from mice from different treatment groups and analyzed for claudin expression levels, and the results are shown in FIG. 1F. As can be seen from fig. 1F: in heart, brain and lung, the expression level of the claudin of the model group is obviously reduced compared with that of a normal mouse, and the expression level of the group treated by MIA602 is obviously recovered. This indicates that: MIA602 is able to maintain the barrier function of blood vessels by reducing the destruction of tight junction proteins in hyperglycaemia-induced vascular lesions.

Example 2: effects of GHRH antagonists on body weight, cardiac contractile function and motor capacity the "CTRL" group, "STZ + MIA602" group and "STZ + vehicle" group of this example were the same as in example 1.

The body weights of mice in different treatment groups at different time points were measured, and the results are shown in fig. 2A. As can be seen from fig. 2A: after one week of STZ molding, the weight of the molding group mice is obviously reduced compared with the normal group; after 4 weeks of treatment with MIA602, the reduced body weight was significantly recovered in the STZ + MIA602 group compared to the STZ + vehicle group, and this trend continued until week 8 of treatment.

Cardiac ultrasound was used to assess contractile function in mice from different treatment groups two months after administration, and the results are shown in figure 2B. As can be seen from fig. 2B: the MIA602 can effectively improve the contractile function of the heart of the hyperglycemic mouse.

The exercise capacity of the mice of the different treatment groups after two months of administration was evaluated, and as shown in fig. 2C and 2D, the exercise duration of the mice of STZ + vehicle group was significantly lower than that of the STZ + MIA602 group. These data indicate that MIA602 can effectively improve the systemic function and state of hyperglycemic mice, maintain normal body weight of mice, and protect systemic vascular function. Further, maintenance of vascular integrity and function by the MIA602 may also reduce hyperglycemia-induced damage to various target organs, such as vascular lesions of the limbs, foot ulcers, eye diseases, and the like.

In addition, blood glucose was measured for mice of different treatment groups at different time points, and the results are shown in fig. 3A; two months after administration, urine glucose levels were measured in mice of different treatment groups, and the results are shown in fig. 3B. As can be seen from fig. 3A and 3B: the GHRH antagonist MIA602 did not affect blood and urine glucose levels in STZ-made mice.

Example 3: effect of GHRH antagonists on vascular endothelial permeability, cellular oxidative stress levels, and claudin primary umbilical vein endothelial cells (HUVECs) were treated with medium at constant sugar (denoted "NG" group), high sugar (33 mM) (denoted "HG" group) to mimic normal and high sugar models, respectively. The HG group was subdivided into a drug administration group (designated "HG + MIA 602") and a control group (designated "HG + vehicle"), and the following treatments were used: respectively use and contain 10 ^-8 Cells were treated after preparation of mol/mL MIA602 or high sugar medium containing equal concentrations of vehicle. The medium of the "NG" group contained equal concentrations of vehicle (denoted as "NG" group or "NG + vehicle" group). After 48 hours of treatment of the different groups of cells, the following different functions were tested:

the generation of ROS was detected using flow-based assay for DCFH-A fluorescent probe-labeled cells, and the results are shown in FIG. 4A. As can be seen from fig. 4A: the ROS generated by the high-sugar modular cells is obviously increased; and the MIA602 can effectively reduce the generation of ROS and improve the stress level.

In FITC-dextran penetration in endothelial cell layer experiments, after treating HUVEC layer in Transwell chamber with different media for 48 hours, respectively, 100 μ L of FITC labeled dextran 10mg/mL was added to the upper Transwell chamber, 50 μ L of lower chamber media was taken at the specified time point and OD was measured on a multifunctional microplate reader to reflect the amount of dextran migrated into the lower chamber, the experimental procedure and results are shown in FIG. 4B. As can be seen from fig. 4B: hyperglycosemia increases permeability of the vascular endothelial cell layer, and MIA602 effectively ameliorates permeability disruption.

Tight junction proteins critical to endothelial cell permeability were further examined in different treatment groups and it can be seen from the immunofluorescent staining results shown in figure 4C: in the HG treatment group, compared with the NG treatment group, the expression levels of intercellular tight junction proteins CLDN5 (green marker) and ZO-1 (red marker) are obviously reduced, while the MIA602 can effectively protect the damage of tight junction (the quantity of red fluorescence and green fluorescence is difficult to accurately reflect due to the fact that colors cannot exist in the drawing of the specification, and can be obviously seen in a colored original drawing). The Western Blot assay data also confirmed this conclusion (FIG. 4D).

Example 4: effect of GHRH antagonists on GHRHR expression levels

The packet setting and processing in this embodiment are the same as in embodiment 3.

Western Blot analysis comparing the expression of GHRHR protein in HUVEC cells of control group and high saccharide group, FIG. 5A shows that: the expression level of GHRHR was significantly increased in the HG group (i.e., the "HG + vehicle" group in example 3) compared to the NG group.

The results of the detection of PKC/P65 phosphorylation levels in HUVEC cells of different treatment groups are shown in FIG. 5B, from which it can be seen that: compared with the NG group, the HG group treated cells have obviously increased phosphorylation levels of PKC and P65, and the MIA602 can effectively inhibit the phosphorylation level.

The Western Blot result of using siRNA to knock down NF kappa B in HUVEC cells is shown in FIG. 5C, and the expression level change of GHRHR is obviously reduced while the expression level of NF kappa B in the HUVEC cells knocked down by NF kappa B-siRNA is reduced. The simultaneous use of Dual Luciferase Reporter (Dual-Luciferase Reporter) experiments verified that NF-. Kappa.B is a transcription factor of GHRHR (shown in FIG. 5D).

From the above results, it is estimated that: the GHRH antagonist effectively inhibits a series of reactions such as hyperglycemia-induced oxidative stress and the like, and further inhibits a downstream NF kappa B pathway, so that the transcription level of GHRHR is reduced, and the protein expression of GHRHR caused by hyperglycemia is reduced.

Example 5: effect of GHRH antagonists on cAMP/PKC/CREB pathway phosphorylation levels and matrix Metalloproteinase expression levels

To clarify the specific mechanism of the damage of vascular endothelial cell claudin by MIA602 to protect high sugars, transcriptomic sequencing was performed on endothelial cells treated differently with vehicle and MIA602 after high sugar modeling, and the results are shown in fig. 6A. Performing KEGG-pathway enrichment analysis on two groups of differential genes according to a sequencing result, screening a PKC/CREB classical pathway with a top-ranked correlation by combining with a GHRHR correlation pathway, and designing a correlation experiment for verification, wherein the method comprises the following specific steps:

the detection of PKC/P65 phosphorylation levels by Western Blot in HUVEC cells from different treatment groups indicated that: compared with the normal sugar group, the phosphorylation levels of PKC and CREB of the cells are obviously increased after the high sugar group is treated for 12 hours, and the MIA602 intervention group can effectively inhibit the phosphorylation level. This indicates that the protective effect of MIA602 on vascular endothelial cells in the hyperglycemic model is related to the PKC/CREB pathway.

The MMP enzyme activity of HUVEC cells of different treatment groups was detected by gelatinase spectrometry, and the result (shown in FIG. 6C) shows that: the activity of endothelial cell matrix metalloproteinases (MMP 2, MMP 9) was significantly increased after high sugar treatment, while MIA602 was able to significantly reduce the increase in MMP activity caused by high sugar treatment.

The expression levels of MMP and tight junction related proteins of HUVEC cells of different treatment groups are detected by Western Blot, and the result (shown in FIG. 6D) also shows that: MIA602 can effectively reduce the high carbohydrate-induced MMP2 and MMP9 protein content, thereby reducing the hydrolysis of tight junction proteins.

From the above experimental results, it is estimated that: GHRH antagonists are used to protect endothelial barrier and vascular integrity by inhibiting cAMP/PKC/CREB pathway phosphorylation, blocking CREB from initiating matrix metalloproteinase transcription, and reducing matrix metalloproteinase hydrolysis on tight junction related proteins.

The raw materials and equipment used in the invention are common raw materials and equipment in the field if not specified; the methods used in the present invention are conventional in the art unless otherwise specified.

The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention, and all simple modifications, alterations and equivalents of the above embodiments according to the technical spirit of the present invention are still within the protection scope of the technical solution of the present invention.

Claims

1. Use of a ghrelin antagonist in the manufacture of a medicament for the prevention or treatment of vasculopathy.

2. The use of claim 1, wherein the vascular disorder comprises vascular endothelial injury.

3. The use of claim 2, wherein said vascular disorder comprises a decrease in vascular endothelial tight junction protein.

4. The use of claim 2, wherein the vascular disorder comprises vascular endothelial cell oxidative stress.

5. The use according to any one of claims 1 to 4, wherein the vasculopathy is a vasculopathy caused by hyperglycemia.

6. The use according to claims 1 to 4, wherein the medicament comprises the following (a) and/or (b):

(a) A growth hormone releasing hormone antagonist;

7. The use of claim 6, wherein the medicament further comprises one or more of a pharmaceutically acceptable carrier, excipient, solvent and buffer.

8. Use of a growth hormone antagonist for the manufacture of a medicament for the prevention or treatment of body or organ damage caused by hyperglycemia.

9. The use of claim 8, wherein the injury to the body or organ is caused by a vascular disorder.

10. The use of claim 8 or 9, wherein the organ comprises a heart, brain, lung or peripheral limb.