Application of GPR146 in treatment of abdominal aortic aneurysm

Abstract

The invention provides an application of GPR146 in treatment of abdominal aortic aneurysm. The application proves that the morbidity and mortality of the aortic aneurysm can be effectively reduced by knocking down or knocking out the GPR146; reducing the diameter of the abdominal aorta; the conditions of artery lumen stenosis, vascular structure disorder, remodeling and elastic fiber fracture are reduced. The application provides a new treatment approach beyond surgery for the clinical problem of abdominal aortic aneurysm treatment.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and specifically provides an application of GPR146 in the treatment of abdominal aortic aneurysm. BACKGROUND

[0002] Abdominal aortic aneurysm is a serious cardiovascular disease, which refers to the abnormal expansion of a certain segment of the abdominal aorta below the renal artery, forming a tumor-like bulge and the diameter exceeding 1.5 times the normal abdominal aortic diameter. Abdominal aortic aneurysm accounts for 3 / 4 of aortic aneurysm, and the incidence is occult. The clinical prevention and treatment faces great challenges. However, there is no clear treatment drug for aortic aneurysm at present, and the postoperative mortality and complications of traditional open surgery are high. The commonly used endovascular repair also has adverse prognosis such as high secondary surgery rate in patients with female, advanced age, poor anatomic conditions of aortic aneurysm, and so on. Therefore, it is of great clinical significance and social value to further study the pathogenesis of abdominal aortic aneurysm, find new intervention targets and develop more effective intervention means for the prevention and treatment of abdominal aortic aneurysm.

[0003] G-protein-coupled receptors (GPCRs) are the largest superfamily of cell membrane receptors in the human body, containing more than 800 members, and are expressed in various organ systems. Abnormal activation of GPCRs can lead to hypertension, vascular dysfunction and even abdominal aortic aneurysm. GPR146 is a classic GPCR. Studies have found that GPR146 can affect blood lipid levels by regulating the secretion of very low density lipoprotein, and then affect the size of aortic atherosclerotic plaques; hypoxia can up-regulate the level of GPR146, and then induce pyroptosis and inflammatory response, causing pulmonary artery endothelial injury and vascular remodeling, and promoting the progression of pulmonary arterial hypertension. It can be seen that GPR146 plays an important role in the cardiovascular system, but the role of GPR146 in abdominal aortic aneurysm has not been reported. SUMMARY

[0004] In one aspect, the present application provides an application of GPR146 in the treatment of abdominal aortic aneurysm.

[0005] In another aspect, the present application provides an application of a reagent for knocking down or knocking out the expression of GPR146 in the preparation of a drug for preventing or treating abdominal aortic aneurysm.

[0006] Further, the reagent for knocking down or knocking out the expression of GPR146 is siRNA, dsRNA, antisense RNA or shRNA against GPR146.

[0007] Further, the reagent for knocking down or knocking out the expression of GPR146 is a CRISPR-Cas9 reagent, a TALENs reagent or a ZFNs reagent for knocking down or knocking out GPR146.

[0008] Further, the reagent for knocking down or knocking out the expression of GPR146 is a reagent for knocking down or knocking out the GPR146 gene shown in Genbank NM_001038703.4 and NM_001303473.2.

[0009] Further, the reagent for knocking down or knocking out the expression of GPR146 is a CRISPR-Cas9 reagent.

[0010] Further, the CRISPR-Cas9 reagent comprises sgRNA with sequences of SEQ ID NO. 1 and SEQ ID NO. 2.

[0011] Further, the CRISPR-Cas9 reagent comprises a Cre-LoxP system reagent.

[0012] Further, the Cre-LoxP system reagent is used for inserting LoxP sites at both ends of the second exon of GPR146 and recombination processing using Cre recombinase.

[0013] Further, the sequence of GPR146-Loxp structure formed after inserting LoxP sites at both ends of the second exon of GPR146 is shown in SEQ ID NO. 4.

[0014] Further, the drug is an oral or injection drug.

[0015] Further, the drug is an injection drug.

[0016] Further, the drug further comprises a pharmaceutically acceptable carrier.

[0017] Further, the drug has one or more of the following effects:

[0018] (1) reducing the incidence of aortic aneurysm;

[0019] (2) reducing the mortality rate caused by aortic aneurysm;

[0020] (3) reducing the diameter of the abdominal aorta;

[0021] (4) reducing the stenosis of arterial lumen, vascular structure disorder, remodeling and elastic fiber rupture.

[0022] The drug can be used in mammals, such as humans or mice.

[0023] The GPR146 gene described in the present application is not limited to the one used in the examples, other GPR146 genes recorded in Genbank can also be used, and those skilled in the art can use these genes to design knockdown or knockout reagents.

[0024] Those skilled in the art can design siRNA, dsRNA, antisense RNA, shRNA, and CRISPR-Cas9 reagents, TALENs reagents, or ZFNs reagents according to means known in the art.

[0025] The present application proves that knocking down or knocking out GPR146 can effectively reduce the incidence and mortality of aortic aneurysm; reduce the diameter of the abdominal aorta; reduce the stenosis of the arterial lumen, vascular structural disorder, remodeling, and elastic fiber rupture. The present application provides a new treatment approach other than surgery for the clinical problem of abdominal aortic aneurysm treatment. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 Schematic diagram of the construction process for GPR146 knock-in mice;

[0027] Figure 2 Schematic diagram of the construction process for GPR146 knockout mice;

[0028] Figure 3 It is shown that the expression of GPR146 in the abdominal aorta of the mouse model of abdominal aortic aneurysm constructed using PCSK9 / AngII is significantly increased;

[0029] Figure 4 The abdominal aortic aneurysm incidence, survival rate, aortic diameter, and elastic fiber staining detection results of GPR146 knock-in and control group mice after constructing the abdominal aortic aneurysm model using the PCSK9 / AngII method.

[0030] Figure 5 The abdominal aortic aneurysm incidence, survival rate, aortic diameter, and elastic fiber staining detection results of GPR146 knock-in and control group mice after constructing the abdominal aortic aneurysm model using the BAPN / AngII method.

[0031] Figure 6 The abdominal aortic aneurysm incidence, survival rate, aortic diameter, and elastic fiber staining detection results of GPR146 knockout and control group mice after constructing the abdominal aortic aneurysm model using the PCSK9 / AngII method.

[0032] Figure 7 The abdominal aortic aneurysm incidence, survival rate, aortic diameter, and elastic fiber staining detection results of GPR146 knockout and control group mice after constructing the abdominal aortic aneurysm model using the BAPN / AngII method. DETAILED DESCRIPTION

[0033] Experimental animals and cells

[0034] Experimental animals and feeding:

[0035] 8-week-old, 20-30 g, using CRISPR / Cas9 technology to construct GPR146 gene mice in C57BL / 6 background (purchased from Jisui Yaoke Biotechnology (Beijing) Co., Ltd.).

[0036] Genes and proteins:

[0037] The GPR146 gene targeted in this experiment is the mouse (Mus musculus) Gpr146 gene, GenBank number NM_001038703.4 (nucleotide sequence), encoding G protein-coupled receptor 146 (NCBI Reference Sequence: NP_001033792.1). Human GPR146 gene (NM_001303473.2) and protein sequence (NP_001290402.1) and mouse have high homology, especially in the ligand binding domain and transmembrane region Key functional regions are highly consistent. Therefore, the research results of mouse models can provide reliable reference for the functional mechanism of human GPR146.

[0038] Specific construction strategy:

[0039] As shown in Figure 1 , the CAG-LoxP-stop cassettle-LoxP-GPR146 CDS sequence is inserted into the H11 site by CRISPR / Cas9-mediated genome editing, and then crossed with SM22α / Tagln-Cre mice to produce VSMCs-specific GPR146 knock-in mice (GPR146 Cre / + ).

[0040] As shown in Figure 2GPR146-Loxp mice were constructed using CRISPR / Cas9-mediated gene editing, as shown in the following: gRNA was designed and transcribed in vitro (see SEQ ID NO. 1 and SEQ ID NO. 2 for details), and a homologous recombination vector (Donor vector) was constructed. Cas9, gRNA, and Donor vector were injected into the zygote of a mouse. Cas9 protein binds to the target site under the guidance of gRNA, causing a double-stranded DNA break. The Donor vector repairs the broken double strand through homologous recombination, thereby inserting two Loxp sites into the 2nd exon of the GPR146 gene, respectively, to construct GPR146-Loxp mice (SEQ ID NO. 1). Using the Cre-Loxp system, Tagln-Cre mice were crossed with GPR146-Loxp homozygous mice to obtain Cre-positive GPR146-Loxp mice, i.e., smooth muscle-specific GPR146 knockout mice (GPR146 SMC- / - ).

[0041] sgRNA sequence

[0042] gRNA No. gRNA Sequence (5'-3') PAM gRNA1 CACCGTACCCTGAGAGCG (SEQ ID NO. 1) GGG gRNA2 CTAAGGGATTATATGG (SEQ ID NO. 2) TGG

[0043] GPR146-Loxp and its surrounding sequence (SEQ ID NO. 3):

[0044]

[0045]

[0046]

[0047] The underlined part represents the Loxp site, the double underlined part represents the Exon2 position, and the knockout content is between the two Loxp sites.

[0048] GPR146-Loxp (SEQ ID NO. 4)

[0049]

[0050]

[0051]

[0052] Experimental cells:

[0053] Human aortic smooth muscle cell line (HASMCs) was purchased from ScienCell Research Laboratories and cultured in smooth muscle cell medium with smooth muscle growth supplement, penicillin-streptomycin, and 2% fetal bovine serum. Cells in passage 3-10 were used for experiments.

[0054] Example 2: Construction of abdominal aortic aneurysm animal model

[0055] Method 1: Construction of abdominal aortic aneurysm model using BAPN and AngII

[0056] Subcutaneous implantation was performed using an AngII osmotic pump. The required AngII dose for each mouse was 1000 ng / kg / min, and the required amount of AngII powder was calculated and added to an appropriate amount of normal saline, which was slowly shaken to dissolve completely. The dissolved AngII drug was injected into the injection hole of the ALZET 2004 osmotic pump. Care should be taken to avoid the generation of air bubbles during injection. The osmotic pump was inverted in a centrifuge tube containing sterile normal saline and incubated in a 37°C water bath overnight. The ALZET 2004 osmotic pump was implanted into the subcutaneous adipose tissue of the mouse. Care should be taken to ensure that the mouse is not infected. After 30 minutes of observation, the mouse was observed for any signs of discomfort before being released.

[0057] Each mouse (about 8 weeks old) consumed about 6-8 ml of water per day. The BAPN dose for each mouse was 200 mg / kg / d. On the day of pump implantation, an appropriate amount of BAPN powder was weighed and dissolved in sterile ddH2O. The solution was used as drinking water for 28 days. A new solution was prepared daily, and the drinking bottle was wrapped in tin foil (BAPN is easily decomposed by light).

[0058] After 28 days, the intervention was completed, and all mice were subjected to tissue sampling. If a mouse died during the model induction period, a necropsy was performed immediately to determine the cause of death. The mouse was observed and recorded.

[0059] Method 2: Construction of abdominal aortic aneurysm model using PCSK9 and AngII

[0060] PCSK9 adeno-associated virus (rAAV8—HCRApoE / hAAT-D377Y-mPCSK9) was purchased from Weizhen Biotechnology (AV208001-AV8). Each mouse was injected with 5*10 11Vg, after the virus is thawed at 4°C in an ice box, it is dissolved in 200 μl of sterile normal saline for tail vein injection. The mouse tail is disinfected with 75% alcohol, and the excess hair on the tail is scraped off to make the blood vessels full. The mouse body is fixed using a visual tail vein injection fixer, the right hand index finger and thumb fix the mouse tail, a disposable 1 ml insulin syringe is used to suck the PCSK9 solution, the needle tip is inclined upward, the needle and blood vessel are at an angle of about 30°, and the needle is gently pricked into the skin immediately after the needle is inserted into the blood vessel, the needle is gently shaken to determine that the needle is in the blood vessel, and the drug solution is slowly pushed. The mice successfully injected with PCSK9 virus solution are returned to the cage and start to be fed with high-fat feed (D12109C).

[0061] Two weeks after the injection of the adeno-associated virus, a subcutaneous implantation is performed using an AngII osmotic pump, and the required AngII dose for each mouse is 1000 ng / kg / min. The required amount of AngII powder is calculated and added to an appropriate amount of normal saline, and slowly oscillated to fully dissolve. The dissolved AngII drug is injected into the injection hole of the ALZET 2004 osmotic pump. Avoid generating bubbles when injecting. The osmotic pump is inverted in a centrifuge tube containing sterile normal saline and incubated in a 37°C water bath overnight. The ALZET 2004 osmotic pump is buried in the subcutaneous adipose tissue of the mouse. Pay attention to disinfection and observation to ensure that the mouse is not infected. After 30 minutes of observation, the mouse is observed for no obvious discomfort before leaving.

[0062] After 28 days, the intervention is over, and all mice are taken. During the model induction period, if a mouse dies, necropsy should be performed immediately, and the cause of death should be observed and recorded.

[0063] Example 3 related detection method

[0064] Aorta sampling and fixation:

[0065] Vessel sampling: perfusion with normal saline and pre-cooled 4% paraformaldehyde (PFA) solution for fixation. After the mouse is anesthetized with 1.25% alverine, the heart, full-length aorta, renal artery, and kidney are separated and photographed.

[0066] Vessel fixation: place the vessel in 4% PFA fixing solution and fix at room temperature for 12 hours. Dehydrate and section for embedding for subsequent staining and other experiments.

[0067] HE staining:

[0068] Paraffin sections were removed, xylene was used to remove paraffin and make the tissue sections transparent. Dehydration was performed with absolute ethanol, 95% ethanol, 70% ethanol and distilled water, respectively. The tissue sections were stained in hematoxylin solution for 5 minutes and in eosin solution for 3 minutes, and then dehydrated and transparentized with 95% ethanol, absolute ethanol and xylene, respectively. Finally, the tissue sections were immersed in xylene for 2-3 minutes. The tissue sections were closed with mounting medium, and then observed and photographed under a microscope.

[0069] Elastic fiber EVG staining:

[0070] The sections were routinely baked at 68°C for 1 h and deparaffinated to water. Verhoeff's staining solution was used to warm room for 1-3 min until the color was dark black, and then rinsed with tap water for 1 min. The sections were differentiated with 2% ferric trichloride solution for 10-20 s, and then observed under a microscope. The elastic fibers were black and the background was gray. The sections were then rinsed with tap water. Van Gieson's solution was used for restaining for 10-15 s, and then dehydrated with absolute ethanol. The sections were transparentized in xylene. The tissue sections were closed with mounting medium, and then observed and photographed under a microscope.

[0071] Immunohistochemical technique:

[0072] The sections were routinely baked at 68°C for 1 h and deparaffinated to water. The sections were boiled in a pressure cooker with EDTA antigen retrieval solution (pH = 9.0) for 1.5 min, and then washed with PBS for 3 times, 5 min each time. Hydrogen peroxide blocking agent (Zhijin Bridge, PV-6002) was added to the tissue to block endogenous hydrogen peroxidase, and then the sections were treated at room temperature for 10 min. The sections were washed with PBS for 3 times, 5 min each time. The sections were blocked with 1% bovine serum albumin for 1 h at room temperature. GPR146 primary antibody (ab117104) was added to the sections, and then the sections were incubated at 4°C overnight. The sections were washed with PBS for 3 times, 5 min each time, and then secondary antibody (HRP labeled goat anti-mouse / rabbit IgG polymer, Zhijin Bridge) was added to the sections, and then the sections were incubated at room temperature for 20 min. The sections were washed with PBS for 3 times, 5 min each time. The sections were developed with freshly prepared DAB developing solution, and then the development was controlled under a microscope at room temperature. The development was stopped with tap water in time. The nuclei were stained with hematoxylin for 1-1.5 min, and then washed with tap water for 1 min. The sections were differentiated with 1% hydrochloric acid for 2 s, and then washed with tap water for 1 min. The sections were returned to blue with blue returning solution (ammonia type) for 5 s, and then washed with running water for 1 min. The sections were dehydrated and transparentized with 95% ethanol, absolute ethanol and xylene, respectively. Finally, the sections were immersed in xylene for 2-3 minutes. The tissue sections were closed with mounting medium, and then observed and photographed under a microscope.

[0073] Example 4 experimental results

[0074] Model construction:

[0075] A mouse model of abdominal aortic aneurysm was constructed using PCSK9 / AngII, with a control group receiving saline. The mouse abdominal aorta was isolated, and GPR146 expression was detected by immunohistochemistry. Figure 3 As shown, compared with the control group, the expression of GPR146 in the abdominal aorta of mice in the PCSK9 / AngII model group was significantly increased.

[0076] like Figure 4 As shown:

[0077] Type (GPR146) into GPR146 Cre / + ) and control (GPR146) + / + Mice in the ) group were given the PCSK9 / AngII method to construct an abdominal aortic aneurysm model, and the heart, aortic length, and renal artery were isolated. Figure 4 Part A of the study found that the incidence of abdominal aortic aneurysms was higher in the GPR146 knock-in group mice compared to the control group (11 / 18 vs 6 / 18). Figure 4 Part B); Survival curves during modeling revealed that the GPR146 knock-in group mice had a worse survival rate. Figure 4 Part C). Aortic ultrasound showed that the abdominal aorta diameter was larger in the GPR146 knock-in group mice (part C). Figure 4 Part D). After separating the aorta, H&E and elastic fiber (Verhoeff's Van Gieson, EVG) staining indicated that GPR146 knock-in mice exhibited more severe arterial stenosis, vascular structural disorder, remodeling, and elastic fiber rupture. Figure 4 (E part). The above results indicate that GPR146 knock-in promotes abdominal aortic aneurysm formation in the PCSK9 / AngII model.

[0078] like Figure 5 As shown:

[0079] Type (GPR146) into GPR146 Cre / + ) and control group (GPR146) + / + Mice were given β-aminopropionitrile (BAPN) / AngII to construct an abdominal aortic aneurysm model. The heart, aortic length, and renal artery were then isolated. Figure 5 Part A of the study found that the GPR146 knock-in mice had a higher incidence of abdominal aortic aneurysms compared to the control group (15 / 20 vs 10 / 20). Figure 5 Part B); Survival curves during modeling revealed that the GPR146 knock-in group mice had a worse survival rate. Figure 5 Part C). Aortic ultrasound showed that the abdominal aorta diameter was larger in the GPR146 knock-in group mice (part C). Figure 5Part D). H&E and EVG staining after aortic dissection indicated that GPR146 knock-in mice exhibited more severe luminal stenosis, vascular structural disorder, remodeling, and elastic fiber rupture. Figure 5 (Part E of the above results) These results further confirm that GPR146 promotes the pathogenesis of abdominal aortic aneurysms.

[0080] like Figure 6 As shown:

[0081] GPR146 knockout (GPR146 SMC / - ) and control group (GPR146) fl / fl A mouse model of abdominal aortic aneurysm was constructed using the PCSK9 / AngII method, and the heart, aortic length, and renal artery were isolated. Figure 6 Part A of the study found that the GPR146 knockout group mice had a lower incidence of abdominal aortic aneurysm compared to the control group (3 / 15 vs 8 / 15). Figure 6 Part B); Survival curves during modeling revealed that the GPR146 knockout group had better survival rates. Figure 6 Part C). Aortic ultrasound showed that the abdominal aortic diameter was smaller in the GPR146 knockout group mice (part C). Figure 6 Part D). H&E and EVG staining after aortic dissection indicated that control mice, compared to GPR146 knockout mice, exhibited more severe luminal stenosis, vascular structural disorder, remodeling, and elastic fiber rupture. Figure 6 (Part E). The above results indicate that knocking out GPR146 can alleviate the incidence of abdominal aortic aneurysms.

[0082] like Figure 7 As shown:

[0083] GPR146 knockout (GPR146 SMC / - ) and control group (GPR146) fl / fl Mice were given the BAPN / AngII method to construct an abdominal aortic aneurysm model, and the heart, aortic length, and renal artery were isolated. Figure 7 Part A of the study found that the GPR146 knockout group mice had a lower incidence of abdominal aortic aneurysm compared to the control group (6 / 15 vs 10 / 15). Figure 7 Part B); Survival curves during modeling revealed that the GPR146 knockout group had better survival rates. Figure 7 Part C). Aortic ultrasound showed that the abdominal aortic diameter was smaller in the GPR146 knockout group mice (part C). Figure 7 Part D). Hematoxylin and eosin (H&E) staining and EVG staining after aortic dissection showed that control mice had more severe luminal stenosis, vascular structural disorder, remodeling, and elastic fiber rupture compared to GPR146 knockout mice.Figure 7 (Part E). The above results further demonstrate that GPR146 knockout has a role in alleviating the incidence of abdominal aortic aneurysms.

Claims

1. Application of GPR146 in the treatment of abdominal aortic aneurysm.

2. Application of reagents that knock down or eliminate GPR146 expression in the preparation of drugs for the prevention or treatment of abdominal aortic aneurysms.

3. In the application according to claim 2, the reagent for knocking down or eliminating GPR146 expression is siRNA, dsRNA, antisense RNA or shRNA targeting GPR146.

4. In the application according to claim 2, the reagent for knocking down or eliminating GPR146 expression is a CRISPR-Cas9 reagent, TALENs reagent, or ZFNs reagent for knocking down or eliminating GPR146.

5. The application according to claim 2, wherein the reagent for knocking down or eliminating GPR146 expression is a reagent for knocking down or eliminating the GPR146 gene shown in Genbank NM_001038703.4 and NM_001303473.

2.

6. In the application according to claim 4 or 5, the reagent for knocking down or eliminating GPR146 expression is a CRISPR-Cas9 reagent.

7. The application according to claim 6, wherein the CRISPR-Cas9 reagent comprises sgRNA with sequences SEQ ID NO.1 and SEQ ID NO.

2.

8. The application according to claim 6, wherein the CRISPR-Cas9 reagent comprises Cre-LoxP system reagents.

9. The application according to claim 8, wherein the Cre-LoxP system reagent is used to insert LoxP sites at both ends of the second exon of GPR146 and to recombinate using Cre recombinase.

10. The application according to claim 9, wherein the sequence of the GPR146-Loxp structure formed after inserting LoxP sites at both ends of the second exon of GPR146 is shown in SEQ ID NO.

4.

11. The application according to any one of claims 2-10, wherein the drug is an oral or injectable drug.

12. The application according to claim 11, wherein the drug is an injectable drug.

13. The application according to claim 11 or 12, wherein the medicament further comprises a pharmaceutically acceptable carrier.

14. The application according to any one of claims 2-13, wherein the drug specifically has one or more of the following effects: (1) Reduce the incidence of aortic aneurysm; (2) Reduce mortality caused by aortic aneurysm; (3) Reduce the diameter of the abdominal aorta; (4) Reduce arterial stenosis, vascular structural disorder, remodeling and elastic fiber rupture.

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