CN114848822A - Application of GPR31 inhibitor in preparation of drugs for preventing and treating vascular calcification - Google Patents

Abstract

The GPR31 inhibitor is applied to preparing medicaments for preventing and treating vascular calcification, which is common pathological manifestations of atherosclerosis, hypertension, diabetes, vascular diseases, chronic kidney diseases, aging and the like. The inhibitor realizes the inhibition effect on the GPR31 gene by utilizing siGPR31, and can be used for remarkably treating smooth muscle cell osteogenic differentiation induced by a high phosphate medium, so that the GPR31 inhibitor has a relatively wide application prospect in the treatment of vascular calcification.

Description

Application of GPR31 inhibitor in preparation of drugs for preventing and treating vascular calcification

Technical Field

The invention relates to an application of a GPR31 inhibitor in preparing a medicament for preventing and treating vascular calcification, and belongs to the technical field of medicines.

Background

Vascular calcification (vascular calcification) is a common pathological manifestation of atherosclerosis, hypertension, diabetic vasculopathy, vascular injury, chronic kidney disease, aging and the like, and is mainly manifested by increased stiffness of vascular walls and reduced compliance.

Coronary calcification is a complex, active, partially bone-mineralization-like process. The reasons and mechanisms for the formation of coronary calcification are complex, including hyperphosphatemia, Vascular Smooth Muscle Cell (VSMC) osteogenic transformation, matrix vesicle release, apoptosis, inflammation, oxidative stress, endoplasmic reticulum stress, and the like. At present, the molecular mechanisms responsible for coronary calcification are not fully understood, and in particular the endogenous promoters of coronary calcification are not known. Thus, there is also a lack of effective treatment strategies to deal with calcification.

G protein-coupled receptors (GPCRs) are cell surface receptors that respond to various stimuli to induce signaling pathways through the cell membrane. In humans, GPCRs are widely expressed in the cardiovascular system, immune system, nervous system, etc., and are involved in growth and development and in a variety of pathophysiological processes. Because of their widespread and functional diversity, GPCR family molecules are considered to be the most potential targets for drug development today, with GPCRs being targeted for action in about 20-30% of FDA-approved marketed drugs.

The orphan receptor GPR31 was a member of the G-protein coupled receptor superfamily, first discovered in 1997 by Alessandra Zingoni et al. The GPR31 protein contains 319 amino acids and has the molecular weight of 35KDa, and is highly expressed in platelets, immune cells and various cancer cells, including bladder cancer cells, breast cancer cells, chronic lymphoblastic leukemia cells and the like. GPR31 is reported to be a high affinity GPCR for the bioactive lipid 12- (S) -HETE. More recently, GPR31 has received much attention for its physiological activity in the context of prostate cancer progression and ischemia. GPR31 is being discussed more and more, but there is little research on vascular calcification.

Disclosure of Invention

The invention aims to provide an application of a GPR31 inhibitor in preparing a medicament for preventing and treating vascular calcification.

In order to achieve the above objects and other related objects, the present invention provides the following technical solutions: use of a GPR31 inhibitor for the preparation of a medicament for the prevention and treatment of vascular calcification.

The preferable technical scheme is as follows: the GPR31 inhibitor is at least one of an inhibitor that reduces GPR31mRNA levels, reduces GPR31 protein levels, and reduces GPR31 function.

The preferable technical scheme is as follows: the GPR31 inhibitor is at least one of a GPR31 antisense oligonucleotide, a GPR31 small interfering RNA, a GPR31 short hairpin RNA, a GPR31 monoclonal antibody, a GPR31 small molecule inhibitor, a nucleic acid molecule targeting GPR31 for gene editing and a GPR31 vaccine.

The preferable technical scheme is as follows: the nucleic acid molecule targeting GPR31 for gene editing further comprises an agent for gene editing operations.

The preferable technical scheme is as follows: the GPR31 small interfering RNA has the sequence as follows: forward oligonucleotide: 5'-UUCAGGAGCUCCUAUCGGA-3', respectively; reverse oligonucleotide: 5'-UCCGAUAGGAGCUCCUGAA-3' are provided.

The preferable technical scheme is as follows: the GPR31 inhibitor is a mouse/rabbit derived/humanized anti-human GPR31 antibody.

The preferable technical scheme is as follows: the vascular calcification refers to the common pathological manifestations of atherosclerosis, hypertension, diabetes, vasculopathy, chronic kidney disease and aging.

In order to achieve the above objects and other related objects, the present invention provides the following technical solutions: a medicament for the prevention and treatment of vascular calcification, said medicament comprising the GPR31 inhibitor described above.

The preferable technical scheme is as follows: the medicament further comprises pharmaceutically acceptable auxiliary materials.

Due to the application of the technical scheme, compared with the prior art, the invention has the advantages that:

the inhibitor realizes the inhibition effect on the GPR31 gene by utilizing siGPR31, and can be used for remarkably treating smooth muscle cell osteogenic differentiation induced by a high phosphate medium, so that the GPR31 inhibitor has a relatively wide application prospect in the treatment of vascular calcification.

Drawings

FIG. 1 shows the change in GPR31 expression in control patients and patients with coronary calcification in example one.

FIG. 2 shows GPR31 expression in different cells of example two.

FIG. 3 is the expression of GPR31 in the aortic root and the initial segment of the coronary artery in the control and coronary calcification mouse model of example three.

FIG. 4 is a graph of the four example of the high and high phosphate medium induced HASMC cell calcification with and without siGPR31 treatment, alizarin red staining and changes in expression of the osteogenic differentiation related transcription factors RUNX2 and Osx.

FIG. 5 is an in vitro calcification culture of the thoracic aorta of C57 mice in example five, with or without siGPR31 treatment, alizarin red staining and immunofluorescence of GPR31 and RUNX 2.

Detailed Description

The following description of the embodiments of the present invention is provided for illustrative purposes, and other advantages and effects of the present invention will be apparent to those skilled in the art from the present disclosure.

Please refer to fig. 1-5. It should be understood that the structures, ratios, sizes, and the like shown in the drawings and described in the specification are for illustrative purposes only and are not intended to limit the scope of the present invention. The following examples are provided for a better understanding of the present invention, and are not intended to limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The experimental materials used in the following examples were all purchased from a conventional biochemical reagent store unless otherwise specified.

The first embodiment is as follows: application of GPR31 inhibitor in preparation of drugs for preventing and treating vascular calcification

In the experimental study of the present invention, RNA-seq sequencing of whole blood samples revealed the most significant upregulation of GPR31 in 4 coronary calcification patients compared to 4 control patients. In human carotid atherosclerotic plaques, GPR31 expression was significantly elevated in calcified plaques. The above results indicate that GPR31 may be involved in the development of coronary calcification.

GPR31 expression was detectable in circulating leukocytes from patients with coronary calcification at levels similar to those in monocytes (THP1), macrophages (macrophages), endothelial cells (HUVECs) and smooth muscle cells (HASMC). GPR31 expression was upregulated after induction of calcification in high phosphate medium in HASMC cells, suggesting that upregulation of GPR31 may be closely related to osteogenic differentiation in HASMC.

LDLR ^-/- Mice were fed a high fat diet for 16 weeks and found to have calcification at atherosclerotic plaques at the root of the aorta and the initial segment of the coronary artery, and GPR31 expression was up-regulated in the aorta and coronary artery of high fat-fed mice. It is shown that GPR31 is closely related to the occurrence and development of mouse coronary calcification.

After interfering with GPR31 expression with GPR31 siRNA, the results showed that osteogenic differentiation of smooth muscle cells was significantly inhibited and RUNX2 and Osx expression was almost no longer upregulated by inorganic phosphate.

In the thoracic aorta in vitro culture model, the thoracic aorta calcification was significantly reduced under GPR31 siRNA. Immunofluorescence results after knocking down GPR31 expression showed that high phosphate medium-induced RUNX2 expression was significantly reduced. The above results demonstrate that inhibition of GPR31 expression using GPR31 siRNA inhibits smooth muscle cell osteogenic differentiation.

Based on the above, GPR31 can be used as a therapeutic target for vascular calcification.

According to the invention, the GPR31 inhibitor is an inhibitor for reducing GPR31mRNA level and/or GPR31 protein level and/or GPR31 protein function, and comprises a GPR31 antisense oligonucleotide, GPR31 small interfering RNA (siRNA), GPR31 short hairpin RNA (shRNA), a GPR31 monoclonal antibody, a GPR31 small molecule inhibitor, a nucleic acid molecule targeting GPR31 for gene editing and a GPR31 vaccine.

In one embodiment of the invention, the inhibitor is an siRNA to the mRNA of GPR31 with an interfering targeting sequence of TTCAGGAGCTCCTATCGGA.

The siRNA sequence is as follows: forward oligonucleotide: 5'-UUCAGGAGCUCCUAUCGGA-3', respectively; reverse oligonucleotide: 5'-UCCGAUAGGAGCUCCUGAA-3' are provided.

According to the invention, the medicament further comprises pharmaceutically acceptable auxiliary materials.

The pharmaceutically acceptable excipients are various excipients commonly used or known in the pharmaceutical field, including but not limited to: diluents, binders, antioxidants, pH adjusters, preservatives, lubricants, disintegrants, and the like.

Such diluents are for example: lactose, starch, cellulose derivatives, inorganic calcium salts, sorbitol, and the like. The binder is, for example: starch, gelatin, sodium carboxymethylcellulose, polyvinylpyrrolidone, and the like. The antioxidant is, for example: vitamin E, sodium bisulfite, sodium sulfite, butylated hydroxyanisole, etc. The pH adjusting agent is, for example: hydrochloric acid, sodium hydroxide, citric acid, tartaric acid, Tris, acetic acid, sodium dihydrogen phosphate, disodium hydrogen phosphate, and the like. Such preservatives are, for example: methyl paraben, ethyl paraben, m-cresol, benzalkonium chloride, and the like. The lubricant is, for example: magnesium stearate, aerosil, talc powder and the like. The disintegrating agent

For example: starch, methyl cellulose, xanthan gum, croscarmellose sodium, and the like.

The dosage form of the medicament of the invention can be in the form of oral preparations, such as tablets, capsules, pills, powders, granules, suspensions, syrups and the like; it can also be administered by injection, such as injection solution, powder for injection, etc., by intravenous, intraperitoneal, subcutaneous or intramuscular route. All dosage forms used are well known to those of ordinary skill in the pharmaceutical arts.

The agents of the invention may be administered to a subject by routes known in the art, including, but not limited to, oral, parenteral, subcutaneous, intramuscular, intravenous, intraperitoneal, intrahepatic, intramyocardial, intrarenal, vaginal, rectal, buccal, sublingual, intranasal, transdermal and the like.

The dosage administered will depend on the age, health and weight of the recipient, the type of drug combination, the frequency of treatment, the route of administration, etc. The drug may be administered in a single daily dose, or the total daily dose may be administered in divided doses of two, three or four times daily. The dose may be administered one or more times, and the administration time may range from a single day to several months or longer.

According to the invention, the medicament can also be used in combination with other medicaments capable of improving or inhibiting vascular calcification.

GPR31 expression changes in control patients and coronary calcifications patients.

4 coronary calcification patients and 4 control patients were selected, whole blood samples were collected for RNA-seq sequencing, and 91 genes were found to be up-regulated and 64 genes were found to be down-regulated. Next, we selected genes with log2FC greater than 1.5 and p <0.05 (30) among the up-regulated genes for analysis and found that the changes in GPR31 expression were most pronounced and barely detectable in control samples, whereas expression was up-regulated in coronary calcification patients.

Embedded sections of carotid plaque from atherosclerotic patients were used for alizarin red staining and immunofluorescence to show patient calcification and GPR31 expression at calcified plaques.

Tissue alizarin red staining:

1) the frozen sections of the human carotid plaque are taken out from a refrigerator at the temperature of-20 ℃, and are placed at room temperature for 30min to be thawed.

2) The embedded reagent was removed by soaking in PBS for 30 min.

3) The sections were soaked in 1% alizarin red stain and stained for 30 min.

4) Acid PBS was washed 3 times.

5) And (5) taking a picture under a body type upright microscope.

Tissue immunofluorescence:

1) frozen sections of human carotid artery were thawed at room temperature for 45 min.

2) The embedded reagent was removed by soaking in PBS for 20 min.

3) The membrane is broken by 0.5% Triton X-100, and soaking for 50min (membrane protein is not required to break membrane).

4) Blocking with 1% BSA at room temperature for 3 h.

5) After blocking was complete, the BSA was poured off and inoculated overnight at 4 ℃ using 1% BSA formulated anti-GPR31 antibody.

6) The following day the antibodies were aspirated and washed 3 times with PBS for 6min each.

7) Rhodamine conjugated fluorescent secondary antibodies at a concentration of 0.1% were then formulated with 1% BSA and incubated for 1h at room temperature.

8) The secondary antibody was aspirated and washed 3 times with PBS for 6min each.

9) Nuclei were then stained with 1% DAPI stain for 15 min.

10) The DAPI staining solution was aspirated off and washed 3 times with PBS for 6min each time.

11) And finally, sealing the plate by using an anti-fluorescence quencher and drying the plate in the dark, and detecting the change of the relative fluorescence intensity after drying the plate in the air.

As shown in fig. 1, a, B: 4 cases of patients with coronary heart disease (control group) and patients with coronary heart disease combined coronary calcification are selected, and RNA-seq detection is carried out on whole blood. C, D: patients with severe carotid stenosis who underwent carotid plaque exfoliation were selected for 3 plaques and stained with alizarin red (C) and divided into 2 groups based on the presence or absence of calcification in the plaques. Immunofluorescence detects GPR31 expression (D).

Example two: GPR31 protein expression in different cells.

Extracting the proteins of circulating leukocytes, endothelial cells, monocytes, macrophages, smooth muscle cells and calcified smooth muscle cells of patients with coronary artery calcification, and analyzing the expression of GPR31 in different types of cells by Western blot.

Western blot：

1) After cell treatment, pre-cooled protein lysate (pre-protease inhibitor) was added. The cells are blown and beaten uniformly, transferred into a 1.5mL centrifuge tube, placed on ice for reaction for 5min, fully shaken and then reacted on ice for 5min, centrifuged at 12000rpm for 10min by a 4 ℃ centrifuge, and the supernatant is collected and stored in a refrigerator at minus 20 ℃ for a long time.

2) Protein concentration was determined using BCA method.

3) SDS-polyacrylamide gel electrophoresis (SDS-PAGE): the SDS-PAGE operation steps mainly comprise: preparing polyacrylamide gel, preparing a sample loading system, loading sample, electrophoresis and the like.

4) Film transfer: after the electrophoresis was completed, the gel was removed and the protein sample in the gel was transferred to an NC membrane in a pre-cooled 1 × transfer buffer.

5) And (3) sealing: after the membrane transfer is finished, the NC membrane is sealed in 5% skim milk for 1h at room temperature.

6) Primary antibody incubation: NC membranes were incubated overnight with RUNX2 and GAPDH antibodies in a 4 ℃ refrigerator.

7) And (3) secondary antibody incubation: after recovery of the primary antibody, the membrane was washed 8min × 3 times with PBST. Membranes were incubated in the corresponding secondary antibody for 1h at room temperature.

8) Exposure: after the secondary antibody incubation was finished, the membrane was washed with PBST for 8min × 3 times. The experimental results were obtained by incubating the membranes with ECL chemiluminescent substrate and exposing in the dark.

As shown in FIG. 2, Western blot was used to measure GPR31 expression levels in circulating leukocytes, monocytes (THP1), macrophages (macrophages), endothelial cells (HUVEC), smooth muscle cells (HASMC) and osteoblasts (HASMC for calcification induction) in patients with coronary calcification. P <0.05(n ═ 3).

Example three: expression of GPR31 in aortic root and initial segment of coronary artery in control and coronary calcification mouse models.

LDLR ^-/- Constructing a coronary calcification model by feeding mice with high-fat diet for 16 weeks, collecting aortic root and heart of the mice, positioning aortic root and coronary artery of the mice by frozen sections, staining the aortic root and coronary artery sections with alizarin red, and detecting the calcium deposition condition. GPR31 expression was detected using immunofluorescence. The tissue alizarin red staining and tissue immunofluorescence steps were as described in example one.

As shown in FIG. 3, LDLR ^-/- Mice were fed a high fat diet for 16 weeks, the aortic root and heart of the mice were collected, and frozen sections were used to locate the aortic root and coronary arteries. A: alizarin red staining is carried out on aortic root and coronary section, and calcium deposition is detected. B: immunofluorescence detects GPR31 expression. White arrow: GPR31 positive region.

Example four: high phosphate medium induced HASMC cell calcification, with or without siGPR31 treatment, alizarin red staining and changes in expression of osteogenic differentiation related transcription factors RUNX2 and Osx.

Cells were seeded in 12-well plates, in four groups: (1) a control group; (2) siGPR31 group; (3) a calcification group; (4) calcification and knock-down of GPR31 group. Hyperphosphates were used to induce osteogenic differentiation of HASMCs. The calcification culture medium is prepared from Na ₂ HPO ₄ And NaH ₂ PO ₄ According to the following steps of 1: 1 in DMEM medium containing 2% FBS and 1% double antibody, and the final PO ₄ ^3- Was 3.0mM, and the calcification medium was added when the cell density in the well plate was 80% and replaced with fresh medium every two days. And after four days, removing the culture medium, adding 4% paraformaldehyde for fixing for 45min, dyeing with 1% alizarin red dye solution for 30min, washing with acidic PBS for three times to remove loose color, taking a picture under a body type microscope, adding 10% acetic acid solution into the hole, sucking the supernatant, and measuring the light absorption value at 405nm for relatively quantitatively analyzing the calcium salt deposition change condition.

After four groups of cells are correspondingly treated, cell RNA is extracted and inverted to obtain cDNA, and qPCR is carried out to detect mRNA expression changes of RUNX2 and Osx.

qRT-PCR：

After the medium was aspirated, 400 μ L of total RNA extraction reagent was added, repeatedly blown up with a pipette until no more viscous, and aspirated into a 1.5mL EP tube at a volume ratio of 5: 1 adding trichloromethane, and oscillating and mixing uniformly, wherein the liquid is pink. And then centrifuging at 12000rpm for 12min by using a centrifuge at 4 ℃, wherein the liquid in the tube is divided into three layers, the red lower layer is organic matter, the middle layer is protein and genome DNA, the upper layer is RNA aqueous solution, absorbing the upper aqueous phase into the enzyme-free tube, avoiding absorbing the middle layer as much as possible, adding isopropanol with the same volume to the enzyme-free tube, and settling overnight in a refrigerator at-20 ℃.

Taking out the settled RNA from the refrigerator, performing a cleaning step, sequentially adding 400 μ L of 75% ethanol solution and 100% ethanol solution, centrifuging at 12000rpm and 4 deg.C for 10min to remove organic reagent residue, sucking away 100% ethanol after the final step is completed, air drying in a clean bench for 30min, and adding 50 μ L ddH ₂ Dissolving RNA in O, shaking and mixing. And (3) measuring the RNA concentration by using an ultramicro spectrophotometer for subsequent reverse transcription.

Reverse transcription was performed using a reverse transcription kit to obtain cDNA, and real-time fluorescent quantitation of mRNA expression of the relevant genes RUNX2 and Osx was performed using SYBR dye.

As shown in FIG. 4, HASMC cells were transfected with either control siRNA or GPR31 siRNA, followed by the addition of inorganic phosphate (3mM) to induce osteogenic differentiation for 7 days. Alizarin red staining detects the osteogenic differentiation degree of smooth muscle cells (a). After dyeing, the alizarin red dye is extracted by 10% acetic acid solution, and the absorbance (B) of the extract at 405nm is detected. qPCR measures GPR31mRNA expression (C). The qPCR detected the mRNA expression (D) of the osteogenic differentiation related genes RUNX2 and Osx. P <0.05(n ═ 3).

Example five: in vitro calcification culture of the thoracic aorta of C57BL/6J mice, treatment with or without siGPR31, alizarin red staining and immunofluorescence of GPR31 and RUNX 2.

Thoracic aorta isolation after sacrifice of normally bred C57BL/6J 10 week old male mice:

1) microscopically removing adipose and connective tissue;

2) cutting thoracic aorta into 5mm short segments by using microshearing, placing in a 24-well plate, adding F12 double non-culture medium, and stabilizing overnight;

3) the next day, F12 double no medium was aspirated away for transfection of siGPR 31;

4) sucking away the transfection medium after one day, adding a calcification medium, and replacing the calcification medium once every 2 days;

5) after 12 days, sections were frozen using OCT. The section is used for observing the calcification of the aorta by alizarin red staining.

Staining with alizarin red:

1) taking the frozen section of the arterial ring out of a refrigerator at the temperature of-20 ℃, and standing at room temperature for 30min for thawing;

2) soaking in PBS for 30min to remove the embedded reagent;

3) soaking the slices in 1% alizarin red dye solution for dyeing for 30 min;

4) washing with acidic PBS for 3 times;

5) taking a picture under a body type upright microscope;

6) alizarin red-stained positive areas were counted using Image J.

As shown in FIG. 5, the thoracic aorta of the wild type mouse was isolated and the aorta was cut into about 5mm segments for in vitro culture. Control or GPR31 siRNA was transfected and then osteogenic differentiation was induced for 10 days by the addition of inorganic phosphate (3 mM). Aorta was cryosectioned, stained with alizarin red for vascular calcification (A), and immunofluorescence for GPR31 and RUNX2 expression (B). P <0.05(n ═ 3).

Frozen sections of tissue were used for immunofluorescence assays for the expression of GPR31 and RUNX 2. Immunofluorescence procedures were as described in example one.

In conclusion, the inhibitor realizes the inhibition effect on the GPR31 gene by utilizing siGPR31, and can be used for remarkably treating smooth muscle cell osteogenic differentiation induced by a high phosphate medium, so that the GPR31 inhibitor has a relatively wide application prospect in the treatment of vascular calcification.

The foregoing is illustrative of the preferred embodiment of the present invention and is not to be construed as limiting thereof in any way, and any modifications or variations thereof that fall within the spirit of the invention are intended to be included within the scope thereof.

Claims (9)

1. Use of a GPR31 inhibitor for the preparation of a medicament for the prevention and treatment of vascular calcification.
2. 2. The use of a GPR31 inhibitor for the preparation of a medicament for the prevention and treatment of vascular calcification according to claim 1 characterized in that: the GPR31 inhibitor is at least one of an inhibitor that reduces GPR31mRNA levels, reduces GPR31 protein levels, and reduces GPR31 function.
3. 3. Use of a GPR31 inhibitor for the preparation of a medicament for the prevention and treatment of vascular calcification according to claim 2 characterized in that: the GPR31 inhibitor is at least one of GPR31 antisense oligonucleotide, GPR31 small interfering RNA, GPR31 short hairpin RNA, GPR31 monoclonal antibody, GPR31 small molecule inhibitor, nucleic acid molecule targeting GPR31 for gene editing and GPR31 vaccine.
4. 4. Use of a GPR31 inhibitor for the preparation of a medicament for the prevention and treatment of vascular calcification according to claim 3 characterized in that: the nucleic acid molecule targeting GPR31 for gene editing further comprises an agent for gene editing operations.
5. 5. Use of a GPR31 inhibitor for the preparation of a medicament for the prevention and treatment of vascular calcification according to claim 3 characterized in that: the GPR31 small interfering RNA has the sequence as follows: forward oligonucleotide: 5'-UUCAGGAGCUCCUAUCGGA-3'; reverse oligonucleotide: 5'-UCCGAUAGGAGCUCCUGAA-3' are provided.
6. 6. The use of a GPR31 inhibitor according to claim 3 for the preparation of a medicament for the prevention and treatment of vascular calcification characterized in that: the GPR31 inhibitor is a mouse/rabbit derived/humanized anti-human GPR31 antibody.
7. 7. The use of a GPR31 inhibitor for the preparation of a medicament for the prevention and treatment of vascular calcification according to claim 1 characterized in that: the vascular calcification refers to the common pathological manifestations of atherosclerosis, hypertension, diabetes, vasculopathy, chronic kidney disease and aging.
8. 8. A medicament for preventing and treating vascular calcification, which is characterized in that: a medicament comprising an inhibitor of GPR31 according to any one of claims 1 to 7.
9. 9. The use of the medicine for preventing and treating vascular calcification as claimed in claim 8, wherein: the medicament further comprises pharmaceutically acceptable auxiliary materials.

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