CN110772636B

CN110772636B - Application of GPR31 inhibitor in preparation of medicines for treating cardiac ischemia-reperfusion injury and related diseases

Info

Publication number: CN110772636B
Application number: CN201911134026.4A
Authority: CN
Inventors: 李红良; 张晓晶
Original assignee: Wuhan University WHU
Current assignee: Wuhan sailaiya Biotechnology Co.,Ltd.
Priority date: 2017-08-21
Filing date: 2017-08-21
Publication date: 2021-03-16
Anticipated expiration: 2037-08-21
Also published as: CN110694071B; CN110947003B; CN107362365A; CN110538327B; CN107362365B; WO2019037222A1; CN110694071A; CN110935022A; CN110538327A; CN110772636A; CN110947003A

Abstract

Experimental research shows that the expression level of GPR31 protein is gradually increased along with the prolongation of tissue ischemia time, and a positive correlation exists between the expression level and the severity of tissue ischemia-reperfusion injury. Overexpression of GPR31 can increase activity of liver cells, cardiac muscle cells and kidney cells caused by H/R treatment. In addition, a decrease in GPR31 expression in hepatocytes may inhibit cellular lipid deposition caused by palmitate and oleate stimulation. Overexpression of GPR31 in cardiomyocytes aggravates cardiac hypertrophy due to angiotensin ii. Therefore, GPR31 inhibitors are useful for the preparation of medicaments for the treatment of diseases associated with ischemia reperfusion injury; myocardial hypertrophy and related diseases; metabolic disorders of the liver.

Description

Application of GPR31 inhibitor in preparation of medicines for treating cardiac ischemia-reperfusion injury and related diseases

The application is a divisional application of Chinese patent application 201710719318.9, which is filed on 8/21/2017 and is named as application of a GPR31 inhibitor in pharmacy.

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to application of a GPR31 inhibitor in preparation of a medicine for treating ischemia-reperfusion injury and related diseases thereof, myocardial hypertrophy and related diseases thereof, inflammatory diseases of heart, lipodystrophy, related diseases and the like.

Background

G protein-coupled receptors (GPCRs) are a class of membrane proteins with 7-transmembrane structures, with over 800 family members, the largest class of membrane proteins in the mammalian genome. 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 is a GPCR family molecule, first discovered in 1997 by Alessandra Zingoni et al (Zingoni, A.et al isolation and chromosomal localization of GPR31, a human gene encoding a reactive G protein-coupled receptor. genomics 42,519-523, doi: 10.1006/gene.1997.4754 (1997)). Currently, GPR31 is primarily being studied in the cancer field, while its function in other pathological processes is not yet known. The GPR31 protein comprises 319 amino acids, has a molecular weight of 35KDa, Is highly expressed in platelets, immune cells, and a variety of cancer cells, including bladder cancer cells, breast cancer cells, chronic lymphoblastic leukemia cells, and the like (Feinmark, s.j.et. the Orphan Receptor GPR31 Is the platform let and HUVEC Receptor for 12(S) -hete.blood 114 (2008)). Research has shown that GPR31 is expressed in colon cancer tissues of clinical patients significantly higher than paracancerous normal tissues, and has a positive correlation with cancer cell metastasis rate and a negative correlation with 5-year survival rate (Zou, Y.et. [ Expression and clinical design of G protein-bound receptor 31 in clinical cancer tissue ]. Zhonghua Wei Chang Wai Ke Za Zhi 18, 935 and 940 (2015)). GPR31 can be combined with RAS family molecules such as KRAS, HRAS, NRAS and the like, and regulates the Cell membrane location of KRAS, thereby participating in the generation and development of cancer (Fehrenbacher N, et. the G protein-coupled receptor GPR31 protein membrane association of KRAS [ J ]. J Cell Biol,2017: jcb. 201609096.).

Ischemia-Reperfusion Injury (IRI) is the first concept proposed by Jennings in 1960, and refers to Reperfusion of blood after Ischemia of tissue and organ, which can not only restore the function of tissue and organ, but also aggravate dysfunction and structural damage of tissue and organ. Ischemia reperfusion injury can occur in many vital organs including the heart, liver, lung, kidney, gastrointestinal tract, and the like.

Liver Ischemia Reperfusion Injury (HIRI) is a common pathological process in liver surgery, and is often seen in the pathophysiological processes such as shock, liver surgery requiring blocking of liver blood flow, and liver transplantation. In recent years, with the development of clinical treatment technologies, operations such as liver transplantation, thrombolytic therapy, and hepatic portal block surgery are more and more developed, and despite the continuous improvement of liver protection, surgical skills, and intraoperative monitoring, liver injury caused by ischemia-reperfusion still is a main cause of postoperative organ nonfunctionality, transplantation failure, and even death of patients. After the liver undergoes ischemia reperfusion, liver histiocytes generate a series of metabolic, structural and functional injuries, and are easy to induce liver failure, which is one of the main reasons influencing disease prognosis, operation success rate and patient survival rate.

Acute coronary artery obstructive disease is one of the main lethal causes of the current cardiovascular and cerebrovascular diseases. Although the treatment of bypass surgery, intervention and thrombolysis has been advanced, the mortality rate of patients with acute myocardial infarction is still high, and one important reason is that no effective method for inhibiting ischemia reperfusion injury caused by the restoration of blood flow of ischemic myocardium is available. Coronary thrombolysis, percutaneous coronary angioplasty, intracoronary dilation, and coronary bypass all suffer from myocardial reperfusion injury. The mechanism of myocardial reperfusion injury mainly comprises free radical, calcium overload, neutrophil adhesion, aggregation and exudation mediated by cell adhesion molecules, apoptosis, nitric oxide, a complement system, renin-angiotensin, nuclear factor-kappa B and the like.

The kidney is also a highly perfused organ, sensitive to ischemia as well as ischemia reperfusion. The renal ischemia-reperfusion injury is an important injury link of ischemic acute renal failure and also a restriction factor influencing the early functional recovery of transplanted kidneys in kidney transplantation. There are many factors involved in the ischemia reperfusion injury, and among them, the inflammation cascade and reactive oxygen species oxidative damage caused by ischemia reperfusion are two major factors of concern. The current prevention and treatment strategies for renal ischemia-reperfusion injury mainly comprise: inhibiting leukocyte activation and leukocyte-endothelial cell interaction, neutralizing active oxygen, and resisting endothelin.

The mechanism of ischemic brain injury also involves at least the following aspects: excitotoxicity, peri-infarct depolarization, inflammation, and apoptosis.

Myocardial hypertrophy is the increase in volume and weight of cardiomyocytes produced by the heart to adapt to various stimuli. The pathological changes include myocardial cell hypertrophy, myocardial interstitial cell proliferation, reconstruction of extracellular matrix of heart and other changes, namely myocardial remodeling. The traditional view is that mature myocardial cells terminate differentiation, lose mitotic competence and cannot enter the cell cycle; however, there is evidence to date that both apoptotic and proliferative processes of cardiomyocytes exist during cardiac development and pathology to maintain a steady state level of cardiac function. There are many diseases causing myocardial hypertrophy clinically, such as primary or secondary hypertension, myocardial infarction, valvular disease, congenital heart disease, etc. Although early myocardial hypertrophy is beneficial for maintaining normal cardiac function, since myocardial hypertrophy itself can also increase myocardial oxygen consumption and decrease myocardial compliance, it can lead to heart failure over time and increase the incidence of sudden death. It is currently believed that mechanical and neurohumoral factors induce myocardial hypertrophy, including renin-angiotensin system components, catecholamines, insulin-like growth factors, nitric oxide synthesis systems, and the like. At the cellular and molecular level, the process of cardiomyocyte hypertrophy mainly comprises four links: the presence of a stimulatory signal, transmembrane signaling, immediate early response to gene activation, transformation of a gene expressing a functional or structural protein to an "embryonal" phenotype. Wherein MAPK family signal pathway, Ca²⁺And signal pathways depending on the phosphatidylinositol-3 kinase, a signal pathway mediated by the phosphatidylinositol-3 kinase and a JAK/STAT pathway are found to participate in signal transmission of myocardial hypertrophy, and the pathways are connected by a myriad of threads to form an intricate and complex signal network (Daviny, etc., the research on the molecular mechanism of myocardial hypertrophy, the development of cardiovascular pathology, Vol.30, No.1 in 2009, 47-50).

Liver plays an important role in fat metabolism, and participates in a plurality of important links in the process of lipid metabolism, including the intake and synthesis of fatty acid, the processing, storage, oxidative decomposition and output of lipid. When the amount of fatty acid obtained by the liver exceeds its processing capacity, lipid is deposited in the liver cells in the form of triglyceride, which results in steatosis of liver cells, becoming simple steatosis of liver, and further developing into non-alcoholic steatohepatitis, and some patients can progress into hepatic fibrosis, liver cirrhosis, and even liver cancer (such as liver cancer, the pathogenesis of non-alcoholic fatty liver disease caused by lipid metabolism disorder, the clinical journal of liver and gall disease, volume 31, stage 7 in 2015, 1050-. Similarly, when the level of free fatty acids in blood is increased or the content of intracellular fat is increased, which exceeds the storage capacity of adipose tissue and the oxidation capacity of each tissue to free fatty acids, excessive free fatty acids are deposited in triglyceride form on target tissues of insulin action such as adipose tissue, muscle and liver, and cause insulin resistance, thus causing a series of metabolic disorders and related diseases such as type II diabetes, metabolic syndrome, cardiovascular and cerebrovascular diseases, etc. (Chenjin Seisan et al, research on lipid metabolism disorders and insulin resistance related progress, Chinese Utility medicine, 2008 volume 3, No. 7, 147-.

Disclosure of Invention

Experimental research shows that the expression level of GPR31 protein in a tissue is gradually increased along with the prolongation of tissue ischemia time, and a positive correlation exists between the expression level and the severity of tissue ischemia-reperfusion injury, which indicates that GPR31 is possibly a novel regulatory factor of tissue or organ ischemia-reperfusion injury.

The over-expression of GPR31 can aggravate the activity reduction of liver cells, myocardial cells and kidney cells caused by hypoxia and reoxygenation treatment and promote the inflammatory response of corresponding cells, and the GPR31 can promote the generation and development of ischemia-reperfusion injury of organs such as liver, heart and kidney and other inflammatory responses generated in the organs.

In addition, the reduction of GPR31 expression in the liver cells can inhibit cell lipid deposition caused by stimulation of palmitate and oleic acid, and the result shows that GPR31 is expected to be used as a novel target for regulating fat accumulation of the liver cells and applied to treatment of abnormal fat metabolism and related diseases.

Over-expression of GPR31 in myocardial cells can aggravate myocardial hypertrophy caused by angiotensin II, and the fact that GPR31 can promote the occurrence and development of diseases related to myocardial hypertrophy is suggested.

On the basis, GPR31 can be used as a therapeutic target for ischemia-reperfusion injury and related diseases, myocardial hypertrophy and related diseases, inflammatory diseases of the heart, fat metabolism abnormality and related diseases.

The technical scheme of the invention is as follows:

in a first aspect, the present invention provides the use of a GPR31 inhibitor for the manufacture of a medicament for the treatment of ischemia reperfusion injury and related diseases, cardiac hypertrophy and related diseases, inflammatory diseases of the heart or abnormal fat metabolism and related diseases.

According to the present invention, the ischemia-reperfusion injury and related diseases are selected from hepatic ischemia-reperfusion injury and related diseases, cardiac ischemia-reperfusion injury and related diseases, renal ischemia-reperfusion injury and related diseases, and/or cerebral ischemia-reperfusion injury and related diseases. The ischemia reperfusion injury can be caused by organ transplantation, partial or complete tissue excision, tissue ischemia caused by vascular embolism and other reasons.

Factors that trigger ischemia reperfusion injury of the liver and its associated diseases include, but are not limited to: liver cyst, liver transplantation, thrombolytic therapy, and hepatic portal block surgery.

Factors that contribute to cardiac ischemia reperfusion injury and related diseases include, but are not limited to: myocardial infarction, myocardial infarction recanalization injury, heart transplantation, coronary thrombolysis, percutaneous coronary angioplasty, intracoronary dilatation, coronary artery bypass.

Causes of renal ischemia reperfusion injury and related diseases include, but are not limited to: kidney transplantation, kidney cyst, renal vascular surgery.

Factors that trigger cerebral ischemia reperfusion injury and its related diseases include, but are not limited to: cerebral apoplexy, cerebrovascular surgery, etc.

Preferably, the ischemia reperfusion injury and related diseases are liver ischemia reperfusion injury, heart ischemia reperfusion injury, kidney ischemia reperfusion injury, and/or brain ischemia reperfusion injury.

According to the present invention, the cardiac hypertrophy and related diseases include, but are not limited to: cardiac hypertrophy, heart failure, arrhythmia, arterial embolism, coronary heart disease, angina, heart block and the like.

There are many diseases causing cardiac hypertrophy, such as primary or secondary hypertension, myocardial infarction, valvular disease, congenital heart disease, etc.

Preferably, the cardiac hypertrophy and related diseases are cardiac hypertrophy and heart failure.

Inflammatory diseases of the heart include, but are not limited to: myocarditis, endocarditis.

According to the present invention, the fat metabolism disorder and related diseases include, but are not limited to: insulin resistance, metabolic syndrome, non-alcoholic fatty liver disease, obesity, diabetes, hyperglycemia, hyperlipidemia, etc.

The disease spectrum of non-alcoholic fatty liver disease includes: simple hepatic steatosis, non-alcoholic steatohepatitis, hepatic fibrosis, liver cirrhosis, and liver cancer.

Preferably, the abnormal fat metabolism and related diseases are non-alcoholic fatty liver disease, obesity, hyperlipidemia, insulin resistance, more preferably: simple hepatic steatosis, non-alcoholic steatohepatitis, obesity, hyperlipidemia.

According to the present invention, a GPR31 inhibitor may be an inhibitor that inhibits GPR31 protein activity or protein level, or an inhibitor that inhibits GPR31 mRNA level. The inhibitory activity may be reversible or irreversible.

Inhibitors that inhibit GPR31 protein activity or protein levels include, but are not limited to, antibodies to GPR31, proteins that inhibit GPR31 protein activity or protein levels, polypeptides, enzymes, natural compounds, synthetic compounds, organic matter, inorganic matter. By inhibitors of GPR31 protein activity or protein levels is meant substances that can bind GPR31 but do not produce a biological response upon binding. The inhibitor may block, inhibit or attenuate a response mediated by the agonist and may compete with the agonist for binding to GPR 31.

The inhibitor for inhibiting the mRNA level of GPR31 may be an antisense nucleic acid sequence thereof, siRNA, miRNA, shRNA, dsRNA, or other protein, polypeptide, enzyme, compound capable of inhibiting the mRNA level of GPR 31.

According to the present invention, the antibodies include, but are not limited to, monoclonal antibodies, synthetic antibodies, polyclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies, chimeric antibodies, single chain fv (scFv), including bispecific scFv, single chain antibodies, Fab fragments, F (ab') fragments, disulfide linked fv (sdfv), and epitope binding fragments of any of the above. In particular, antibodies for use in the present invention include immunoglobulin molecules and immunologically active portions of immunoglobulin molecules. The immunoglobulin molecules used in the present invention may be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecule. Preferably, the antibody is a human or humanized monoclonal antibody. As used herein, "human" antibodies include antibodies having the amino acid sequence of a human immunoglobulin, and include antibodies isolated from a human immunoglobulin library or from a mouse or other animal in which antibodies are expressed from human genes.

In one embodiment of the invention, the inhibitor is shRNA of the mRNA of GPR31 interfering with a targeting sequence of CACTCTCCTGCCTTCAGTTTG.

Preferably, the shRNA sequence is: 5'-CCGGCACTCTCC TGCCTTCAGTTTGCTCGAGCAAACTGAAGGCAGGAGAGTGTTT TTG-3' for positive oligonucleotide; reverse oligonucleotide 5'-AATTCAAAAACACTCTCCTGCCTT CAGTTTG CTCGAGCAAACTGAAGGCAGGAGAGTG-3'.

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 disintegrant is, 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 combined with other medicaments which can improve or inhibit diseases related to the ischemia-reperfusion injury.

According to the invention, the medicament can also be combined with other medicaments capable of improving or inhibiting myocardial hypertrophy and related diseases.

According to the present invention, the drug may also be used in combination with other drugs that ameliorate or inhibit inflammatory diseases of the heart.

According to the present invention, the drug may also be used in combination with other drugs that ameliorate or inhibit fat metabolism disorders.

In a second aspect, the invention provides the use of a vector expressing an shRNA targeting the mRNA of GPR31 in the manufacture of a medicament for the treatment of ischemia reperfusion injury and a disease associated therewith, myocardial hypertrophy and a disease associated therewith, an inflammatory disease of the heart, or an abnormality in fat metabolism and a disease associated therewith.

The disease is as defined above.

The shRNA interference targeting sequence is CACTCTCCTGCCTTCAGTTTG.

The vector may be an expression vector. The expression vector may comprise a promoter and a transcription termination sequence operably linked to the shRNA sequence described above.

The expression vector may be a eukaryotic cell expression vector.

The eukaryotic expression vector may be a plasmid expression vector or a viral expression vector.

The plasmid expression vector may be, but is not limited to, pcDNA3.1+/-, pcDNA4/HisMax B, pSecTag 2A, pVAX1, pBudCE4.1, pTracer CMV2, pcDNA3.1(-)/Myc-His A, pcDNA6-Myc/His B, pCEP4, pIRES, pIRESneo, pIRES hyg3, pCMV-Myc, pCMV-HA, pIRES-puro3, pIRES-neo3, pCAGGS, pSilencer1.0, pSilencer2.1-U6 hygro, pSilencer3.1-H1 hygro, pSilencer3.1-H1 neo, and pSilencer4.1-CMV neo.

The viral expression vector may be a lentiviral vector, an adenoviral vector, an adeno-associated viral expression vector or other type of viral vector, including but not limited to pLKO.1, pLVX-IRES-ZsGreen1, pCDH-EF1-Luc2-T2A-tdTomato, pCDH-MSCV-MCS-EF1-Puro, pCDH-MSCV-MCS-EF1-copGFP, pLVX-ZsGreen1-C1, pAdEasy-1, pShuttle-CMV, pShuttle, pAdTrack, pAdTrack-CMV, pShuttle-IRES-hrGFP-1, pShuttle-IRES-hrGFP-2, pShuttle-CMV-lacZ, pShuttle-CMV-EGFP-C, pEGFP 1, pBHGE3, pAAV-pHAV, pAAV-pAVV-pLKO, preferably pLKO.

In a third aspect, the invention provides the use of a lentiviral vector comprising an shRNA targeting the mRNA of GPR31 in the manufacture of a medicament for the treatment of ischemia reperfusion injury and a condition associated therewith, myocardial hypertrophy and a condition associated therewith, an inflammatory condition of the heart, or an abnormal fat metabolism and a condition associated therewith.

The disease is as defined above.

The shRNA interference targeting sequence is CACTCTCCTGCCTTCAGTTTG or other targeting sequences capable of interfering GPR31 expression.

Preferably, the lentiviral vector is a plko.1 vector.

The pharmaceutically acceptable carrier which can be used in the preparation of the medicaments of the second and third aspects may be an injection carrier as is conventional in the art, such as an isotonic NaCl solution, an isotonic glucose solution, or an isotonic solution containing a buffer system, such as a PBS solution, etc. Protective agents for preventing inactivation of lentiviruses by physical or chemical changes, such as divalent cation salts or surfactants, can also be optionally added according to the needs of the preparation.

Drawings

FIG. 1: western-blot assay of the amount of GPR31 protein expression in liver tissues at different ischemic times. GAPDH is shown as a control standard.

FIG. 2: graph identifying the expression of GPR31 protein after transfection of L02 cells by GFP and GPR31 overexpression lentivirus. GAPDH is shown as a control standard.

FIG. 3: statistical plots of LDH release measurements from L02 cells after hypoxic and reoxygenation treatments with over-and normal expression of GPR31, respectively (n.s. for P.gtoreq.0.05, and x.for P < 0.01).

FIG. 4: the L02 cells respectively express GPR31 under the condition of being over-expressed and normally express, and after being treated by hypoxia and reoxygenation, the mRNA content of inflammatory factors Il-6, Tnf-alpha and chemotactic factors Cxcl2 is detected by RT-PCR (the value is 0.01 and is less than or equal to P <0.05, and the value is P < 0.01).

FIG. 5: graph identifying the expression of GPR31 protein of H9C2 cells after transfection of GFP and GPR31 overexpression lentivirus.

FIG. 6: statistical analysis of cell activity of H9C2 cells after hypoxic and reoxygenation treatment under overexpression and normal expression of GPR31, respectively (n.s. represents P.gtoreq.0.05, x represents 0.01. ltoreq. P <0.05, x represents P < 0.01).

FIG. 7: graph identifying GPR31 protein expression of HK2 cells after transfection with GFP and GPR31 overexpression lentiviruses.

FIG. 8: statistical analysis of cellular LDH release measurements after hypoxic and reoxygenation of HK2 cells overexpressing and normally expressing GPR31, respectively (n.s. for P.gtoreq.0.05, x. for P < 0.01).

FIG. 9: graph for identifying the content of GPR31 gene mRNA after shRNA and shGPR31 lentivirus transfection of L02 cells (x represents P < 0.01).

FIG. 10: microscopic image of oil red O staining of hepatocytes after stimulation with Palmitate (PA) and Oleate (OA) (PA 0.5mM + OA 1mM) in the presence of low and normal expression of GPR31 in L02 cells, respectively. "PA + OA" stands for palmitate and oleate stimulated groups.

FIG. 11A: H9C2 cells were cytoscopically imaged after treatment with angiotensin ii in the presence of over-and normal expression of GPR31, respectively.

FIG. 11B: statistical plot of cell surface area of H9C2 cells after treatment with angiotensin ii in the presence of over-and normally expressed GPR31, respectively (n.s. for P ≧ 0.05, for P < 0.01).

FIG. 11C: statistical plots of RT-PCR measurements of mRNA expression levels of the cellular mast marker gene Anp and Myh7 in H9C2 cells treated with angiotensin II under the conditions of over-expression and normal expression of GPR31, respectively (n.s. for P.gtoreq.0.05, and x. for P < 0.01).

Detailed Description

The present invention is further described below with reference to examples. It should be noted that the examples are not intended to limit the scope of the present invention, and those skilled in the art will appreciate that any modifications and variations based on the present invention are within the scope of the present invention.

The chemical reagents used in the following examples are conventional and are commercially available. The experimental methods not specifically described are all the conventional ones known in the art.

The animal models and methods of measurement of various research indices used in the following examples:

experimental animals: wild type mice (purchased from Beijing Huafukang Biotechnology GmbH) of 8-10 weeks old, 24-27 g in weight and male C57BL/6 strain in background are selected as experimental objects.

Animal feeding, all experimental mice are fed in SPF grade experimental animal center of Wuhan university. Feeding conditions are as follows: the room temperature is 22-24 ℃, the humidity is 40-70%, the illumination time is 12h with alternating light and shade, and the drinking water can be freely taken.

HEK293T, human embryonic kidney cells, purchased from chinese academy of sciences cell bank under catalogue number GNHu 43.

L02, human liver cell line, purchased from cell banks of Chinese academy of sciences, catalog number GNHu 6.

H9C2, rat cardiomyocytes, purchased from chinese academy of sciences cell bank under catalogue number GNR 5.

HK2, human renal proximal tubular cells, purchased from the cell Bank of Chinese academy of sciences, Cat. No. SCSP-511.

The cells were all cultured in DMEM high-glucose medium (containing 10% FBS, 1% penicillin-streptomycin). And (3) culture environment: 37 ℃ and 5% CO₂。

Mouse liver ischemia reperfusion (I/R) injury model construction:

1) mice were fasted 12h before surgery and had free access to water.

2) After the mice were successfully anesthetized with 3% sodium pentobarbital before surgery, they were stood flat to immobilize the limbs, the abdominal region of the mice was shaved with a shaver, and the region was sterilized with 10% iodine tincture and 75% ethanol.

3) An incision is made in the middle of the abdomen to expose the hepatic pedicle of the left and middle lobes of the liver.

4) The portal vein and hepatic artery of the middle and left lobes were clamped with a non-invasive vascular clamp to allow approximately 70% of the liver to become ischemic, and after 0.5min, the blocking lobe was seen to whiten compared to the non-blocking right lobe, indicating successful blocking and maintenance of ischemia for 60min (Sham group mice were operated in parallel with surgery group mice, but no blood flow blocking was performed).

5) Removing the vascular clamp after 60min of ischemia, recovering ischemic liver blood flow, closing abdominal cavity, and feeding the mice after operation for observation.

Material taking: the mice of a Sham operation group (Sham) and an ischemia reperfusion group are taken after 1h of operation, 3 percent sodium pentobarbital is used for excessive anesthesia, liver tissues in an ischemic area are taken and immediately placed in liquid nitrogen for more than 30min, and then the liver tissues are stored in a refrigerator at the temperature of 80 ℃ below zero and used for RT-PCR and Western blot analysis.

RT-PCR

1) Extraction of RNA from cells

Collecting cells, washing the cells for 2 times by PBS buffer solution, adding 1ml of TRizol after the washing, blowing the cells uniformly by a sample injector, sucking the cells into a 1.5ml centrifuge tube, shaking the cells by a vortex mixer for 30s, and standing the cells at room temperature for 5min to completely dissociate the nucleoprotein from the nucleic acid.

② centrifuging at 12000r/min at 4 ℃ for 5min, taking supernatant, adding 200 mul chloroform, shaking by a vortex mixer for 30s, and standing on an ice box for 10 min;

③ centrifuging at 12000r/min at 4 ℃ for 15min, taking supernatant, adding 0.5ml of isopropanol, fully and uniformly mixing, standing on an ice box for 10min to ensure that RNA is fully precipitated;

fourthly, centrifuging the mixture for 15min at the temperature of 4 ℃ and the speed of 12000r/min, removing supernatant, adding 1ml of precooled 75% ethanol, and shaking the mixture by a vortex mixer for 30s to wash RNA sediment;

fifthly, centrifuging at 12000r/min at 4 ℃ for 5min, removing supernatant, and quickly air-drying the precipitate. The extracted RNA is dissolved by adding a proper amount of DEPC deionized water.

2) Reverse transcription

Reverse transcription experiments were performed using the Transcriptor First Strand cDNA Synthesis Kit (04896866001, Roche, Basel, Switzerland) reverse transcription Kit according to the Kit instructions.

Western blot

1) Tissue protein extraction

Putting 3-4 steel balls into an EP tube precooled in dry ice, and adding a weighed and quantified tissue sample.

Secondly, PMSF is added into the lysate, mixed evenly and added into the sample, and then shaken up quickly.

Thirdly, grinding the sample in an adapter of a precooling grinder at the temperature of minus 80 ℃, wherein the grinding parameters are set to be 30Hz/s and 90 s.

Fourthly, after the grinding is finished, the steel ball is placed on ice for 10min and taken out.

Cracking the sample (5 KHz/time, 1s each time, 1s interval, repeating 10 times) by using an ultrasonic cracking instrument, and standing on ice for 10min after the ultrasonic is finished.

Sixthly, putting the sample into a pre-cooled centrifuge at 4 ℃, and centrifuging for 30min at 12000 rpm/min.

Seventhly, sucking the supernatant, transferring the supernatant into a new EP tube, and centrifuging the supernatant at 14000rpm/min for 10min at 4 ℃.

Eighthly, sucking the supernatant, transferring the supernatant into a new EP tube, continuing to centrifuge at 4 ℃ and 14000rpm/min for 5 min.

Ninthly, accurately sucking clear liquid and performing Protein quantification by using a BCA Protein Assay Kit (Pierec, 23225).

2) Protein extraction from cells

Adding the cell into a lysis solution, centrifuging after the cell is lysed, taking a supernatant, and quantitatively collecting a Protein sample by using a BCA Protein Assay Kit.

3) Sample loading and electrophoresis

Preparing electrophoresis gel, and adding electrophoresis liquid into an electrophoresis tank. And loading the protein sample into an SDS-PAGE gel loading hole, and starting electrophoresis after the sample application is finished.

4) Rotary film

Firstly, preparing a film transfer liquid, and precooling at 4 ℃.

Soaking the PVDF membrane in methanol for 15s before use, and then putting the PVDF membrane into a membrane transferring solution for later use.

Taking out the gel in the gel plate, washing the gel with a film transfer liquid, and covering the PVDF film on the gel plate.

Fourthly, the transfer membrane voltage is set to be 250V, the current is set to be 0.2A, and the transfer is carried out for 1.5 h.

And fifthly, taking out the PVDF film after the transfer is finished.

5) Sealing of

The protein membrane was placed in a prepared TBST, and the membrane-transfer solution was washed off. Placing the protein membrane in the sealing solution, slowly shaking on a shaking table, and sealing at room temperature for 1-4 h.

6) Primary antibody incubation

(ii) washing the protein membrane 3 times with TBST for 5min each time.

Secondly, sealing the film into the hybrid bag by a sealing machine, adding primary antibody and sealing.

③ put the hybridization bag into a shaker at 4 ℃ overnight.

7) Incubation with secondary antibody

The membrane was taken out and washed 3 times with TBST for 5min each time, and primary antibody was recovered.

② the membrane is put into the corresponding secondary antibody dilution added with secondary antibody, and incubated for 1h in dark.

8) Protein detection

After incubation, wash 3 times with TBST for 5min each. Bands of interest were detected using a Bio-Rad Chemi Doc XRS + gel imaging system.

GPR31 overexpression plasmid construction:

1) GPR31 gene is amplified by PCR, and primers are as follows:

forward direction: 5'-ACACCGGCGGCCACGCGTATGCCATTCCCAAACT GCTC-3', respectively;

and (3) reversing: 5'-GGAGGTACCTCCGGATCCTTACTTATCGTCGTCAT CCTTG-3', respectively;

2) the PCR products were subjected to agarose gel electrophoresis, followed by recovery of DNA fragments using a DNA gel recovery kit (Tiangen);

3) the resulting DNA product is combined with restriction endonucleases Fastdigest restriction enzymes (Thermo),

buffer or

Green buffer、ddH₂O is mixed uniformly (50. mu.l system) and placed at 37 ℃ for reaction. Use of

AxyPrep^TMThe cleavage product was recovered from the PCR Clean-Up Kit (Axygen).

4) Use of

PCR one-step directional cloning kit (Novoprotein), performing recombination reaction according to kit instructions;

5) preparing escherichia coli competent cells, performing a transformation experiment on the ligation product, coating a plate, placing the plate in an incubator at 37 ℃, and culturing overnight;

6) the overnight-cultured plates were removed from the 37 ℃ incubator, colonies were picked, shaken, and colonies were detected for PCR positive clones.

7) The PCR-positive strain was inoculated into 5ml LB medium (containing resistance) in an amount of 5-10. mu.l, and cultured overnight in a shaker at 37 ℃ and 220 rpm.

8) The overnight-cultured bacterial suspension was taken out, and the turbid bacterial suspension was subjected to plasmid extraction (Tiangen plasmid DNA miniprep).

9) The extracted plasmid can be directly used for GPR31 transient transformation or construction of a lentivirus stable cell line.

GPR31 interference plasmid construction

1) A GPR31 targeted interference sequence is CACTCTCCTGCCTTCAGTTTG, and an oligonucleotide suitable for a pLKO.1 vector is designed; forward oligonucleotide 5 'CCGGCACTCTCCTGCCTTCAGTTTGCTCGAGCAAACTGAA GGCAGGAGAGTGTTTTTG 3'; reverse oligonucleotide 5 'AATTCAAAAACACTCTCCTGCCTTCAGTTTGCTCGAGCAAACTG AAGGCAGGAGAGTG 3'; the negative control siRNA sequence was: CAACAAGATGAAGAGCACCAA, respectively;

2) dissolving the two oligonucleotides in sterile water in half to obtain a solution with a final concentration of 100mM, and fusing;

3) performing enzyme digestion reaction, recycling enzyme digestion products, performing ligation reaction, converting, selecting monoclonal antibody and sequencing, and extracting plasmid according to the step of 'construction of GPR31 expression plasmid';

4) the resulting plasmids can be used for lentivirus-mediated construction of GPR31 knockdown cell lines.

Lentiviral vector construction and packaging

1) The 293T cells were trypsinized and counted at 1X 10⁶One 293T/well was transferred to a 6-well plate.

2) Transfection was initiated when the cells were confluent to 80%.

3) A1.5 ml sterile EP tube was taken and 2 packaging plasmids (pSpax and pMD2G) and 1. mu.g each of the over-expression or interference plasmids were added in 100. mu.l of serum-free medium. Gently mix well and incubate for 5min at room temperature.

4) A1.5 ml sterile EP tube was taken and 3. mu.l PEI (1.6. mu.g/. mu.l) was dissolved in 100. mu.l serum free medium. Gently mix well and incubate for 5min at room temperature.

5) The DNA solution and PEI solution were gently mixed. Incubate at room temperature for 15 min.

6) The DNA-PEI mixed solution is added dropwise into a 6-well plate.

7) After 6h of transfection, fresh medium was replaced.

8) Supernatants containing virus were harvested 48-72h after transfection. Centrifugation was carried out at 3000rpm for 10min to remove the precipitate, and filtration was carried out with a 0.45 μm filter.

9) The filtered virus was used immediately for infection or stored at-80 ℃.

Cell hypoxia reoxygenation (H/R):

1) the cells were cultured to log phase, washed 2 times with prewarmed PBS and discarded.

2) Dividing cells into normal control group and H/R experimental group, replacing control group with complete culture medium, placing at 37 deg.C and 5% CO₂Culturing, changing sugar-free serum-free DMEM medium for experimental groups, and placing O₂/CO₂In the incubator of the cell culture system (37 ℃, 5% CO)₂，1％O₂) And (4) carrying out anoxic culture, wherein after different times of anoxic culture, the experimental group is replaced by a complete culture medium for reoxygenation culture.

3) After reoxygenation to the predetermined reoxygenation culture time, the supernatant was discarded, washed 2 times with PBS, and the cells were collected and stored at-80 ℃ for further use.

LDH release and cell viability (cell viability) assay:

LDH release was measured using a colorimetric LDH cytotoxicity assay kit (G1782, Promega, Madison, Wis., USA). Cell activity was measured using a non-radioactive CCK-8 kit (CK 04; Dojindo, Kumamoto, Japan). And carrying out correlation detection according to the instruction.

Dyeing with oil red O:

1) the sample group and the control group were washed 2 times with 1 × PBS, and fixed for 20min by adding 300 μ l of 3% paraformaldehyde;

2) washing with 1 × PBS for 2 times, adding 60% isopropanol, and rinsing for 10 s;

3) washing with 1 × PBS for 2 times, and drying in a fume hood;

4) adding oil red O into 500 mul of each hole for dyeing for 1 h;

5) washing with 1 × PBS for 2 times, sorting with 60% isopropanol, and washing with 1 × PBS for 2 times; and (6) microscopic examination and photographing.

Immunofluorescence staining of myocardial cells:

1) cells were fixed with 4% paraformaldehyde;

2) PBS wash 3 times, each time for 5 min;

3) adding 0.2% Triton, and shaking on shaking table for 5 min;

4) PBS wash 3 times, each time for 5 min;

5) dropwise adding 8% sheep serum, and incubating in a wet box in a shaking table at room temperature for 60min for sealing;

6) discarding the blocking solution, adding dropwise primary antibody (Anti-alpha-actin, Millipore, #05-384) (1% sheep serum 1:100 dilution), and standing at 37 deg.C for 2h or 4 deg.C overnight;

7) discarding the primary antibody, washing with PBS for 4 times, each for 5 min;

8) 30 μ l of diluted secondary antibody (Alexa) was added

488donkey anti-mouse IgG, Invitrogen, A21202, diluted with PBS at a ratio of 1: 200), incubated at 37 ℃ for 60 min;

9) discarding the secondary antibody, washing with PBS for 5 times, each for 5 min;

10) slides were mounted with the DAPI on a SlowFade Gold anti reagent and photographed under a fluoroscope.

Example 1 changes in expression of GPR31 in liver tissue at different times of ischemia

C57 mice were randomly divided into 6 groups, which were Sham group and operation group (divided into 5 different time points: ischemia 5min, 10min, 20min, 40min, 60min), liver tissues of the operation group mice and Sham group mice were taken, and changes in GPR31 protein content in each group of liver tissues were detected by Western blot (3 independent repeat experiments). Wherein the primary antibody used by WB is: Anti-GPCR GPR31 antibodyy (ab 75579; Abcam), secondary antibody: the Peroxidase assay was performed using a Peroxidase goat anti-rabbitt-IgG (H + L) (# 111-. GAPDH is used as a control standard for detecting the expression quantity.

The results are shown in figure 1, with the WB results in Sham group showing almost no GPR31 band, and with the prolongation of ischemia time in the operative group, GPR31 protein band became more and more evident. This result indicates that there is a positive correlation between the expression of GPR31 protein and the severity of hepatic ischemia-reperfusion injury.

Example 2 Effect of GPR31 overexpression on H/R treatment-induced L02 cell injury and inflammatory response

L02 cells were divided into 4 groups: GFP control group, GPR31 control group, GFP H/R group, GPR 31H/R group. Adherent L02 cells were transiently transfected with the corresponding plasmids, respectively, and 24H followed by H/R treatment (6H hypoxia, 6H reoxygenation). After plasmid transfection was complete, total cellular protein was extracted and Western blot was used to detect the overexpression of GPR31 (3 independent replicates each with 3 replicates). Measurement of LDH release (6 replicates per group) in culture media after H/R treatment was completed to evaluate the effect of GPR31 overexpression on H/R-induced hepatocyte injury; RNA was extracted and analyzed by RT-PCR (2 independent replicates, 3 replicates each) to determine the change in mRNA levels of inflammatory-related cytokines and chemokines to assess the effect of GPR31 overexpression on the H/R-induced inflammatory response of hepatocytes. The LDH release detection result is that the GFP control group value is 1, and the ratio of each group to the rest is calculated.

The primer sequences used for RT-RCR are as follows:

gene	Forward primer	Reverse primer
			Il6	TCTGGATTCAATGAGGAGACTTG	GTTGGGTCAGGGGTGGTTAT
Tnfα	TACTCCCAGGTCCTCTTCAAGG	TTGATGGCAGAGAGGAGGTTG
			Cxcl2	GCGCCCAAACCGAAGTCATA	TTTCTTAACCATGGGCGATGC

The results of the GPR31 overexpression WB assay are shown in fig. 2, where GPR31 overexpression histone bands were significantly enhanced compared to the GFP group, i.e., GPR31 overexpression was significant in L02 cells.

The results of the LDH release assay are shown in fig. 3, and there was no significant difference in the LDH release of GPR31 control group compared to GFP control group. When H/R treatment was performed, LDH release was significantly increased, and the release of LDH in GPR31 overexpressed group was significantly increased over that in GFP group. This result suggests that GPR31 overexpression exacerbates H/R treatment-induced hepatocyte injury and hepatotoxicity.

The results of mRNA detection of inflammatory factors and chemokines with actin as a control standard are shown in FIG. 4, and after H/R treatment, the mRNA contents of inflammatory factors Il-6, Tnf-alpha in GPR31 overexpression group and chemokine Cxcl2 are all increased remarkably. This result suggests that GPR31 overexpression exacerbates the H/R treatment-induced inflammatory response of hepatocytes.

Example 3 Effect of GPR31 overexpression on H/R treatment-induced H9C2 cell Activity

H9C2 cells were divided into 4 groups: GFP control group, GPR31 control group, GFP H/R group, GPR 31H/R group. The corresponding recombinant lentivirus virus liquid infects cultured H9C2 cells respectively, and H/R treatment (hypoxia for 1H and reoxygenation for 6H) is carried out after 24H. After plasmid transfection, total cell protein was extracted and Western blot was used to detect over-expression of GPR31 (3 independent replicates). Cell activity was measured after H/R completion (6 replicates per group). The ratio of each of the remaining groups to the group was calculated with the detection result of the GFP control group as 1.

WB assay results as shown in fig. 5, GPR31 overexpression histone bands were significantly enhanced compared to the GFP group, i.e., GPR31 overexpression was significant in H9C2 cells.

The cell activity test results are shown in fig. 6, and the cell activity of GPR31 control group was not significantly different from that of GFP control group. When treated with H/R, the cell activity was significantly reduced compared to the control. When GPR31 is over-expressed, the reduction degree of the cell activity of GPR 31H/R group is obviously larger than that of GFP H/R group. This result indicates that GPR31 overexpression significantly promotes H/R-induced cardiomyocyte damage and decreases cardiomyocyte activity.

Example 4 Effect of GPR31 overexpression on HK2 cell injury following H/R treatment

HK2 cells were divided into 4 groups: GFP control group, GPR31 control group, GFP H/R group, GPR 31H/R group. And respectively infecting the cultured HK2 cells with corresponding lentiviral virus liquid, performing H/R treatment (hypoxia for 3H and reoxygenation for 24H) after 24H, extracting total cell protein after plasmid transfection is finished, and detecting the over-expression condition of GPR31 by Western blot (3 independent repeated experiments). The amount of LDH released in the medium (6 replicates per group) was measured after H/R was completed to evaluate the effect of GPR31 overexpression on H/R-induced renal cell injury. The LDH release detection result of the GFP control group was taken as 1, and the ratio of each of the remaining groups to the group was calculated.

The results of GPR31 overexpression assay in HK2 cells are shown in fig. 7, and the GPR31 protein content in GPR31 overexpressing group cells was significantly increased compared to the GFP group.

The results of the measurement of LDH release amount of kidney cells are shown in FIG. 8, and the LDH release of GPR31 control group is not significantly different from that of GFP control group. When H/R treatment was performed, LDH release was significantly increased, and the release of LDH in GPR31 overexpressed group was significantly increased over that in GFP group. This result suggests that, in contrast to GPR31 acting in liver, cardiomyocytes, overexpression of GPR31 exacerbates H/R-treatment-induced renal cell injury.

Example 5 Effect of GPR31 knockdown on hepatocyte fat deposition

L02 cells were divided into 4 groups: shRNA control group, shGPR31 control group, shRNA experimental group and shGPR31 experimental group. Adherent L02 cells were transiently transfected with the corresponding plasmids, 24h later, and then stimulated with Palmitate (PA) and Oleate (OA) (PA 0.5mM + OA 1mM) in both experimental groups, and the same amount of BSA in the control group, and 12h later, oil red O staining was performed.

The primer sequences used for RT-PCR are as follows:

gene	Forward primer	Reverse primer
			GPR31	TCAACCTGCTGTCTCCTCAG	GTGCTTCCTGCCAGATGATG

After plasmid transfection, cellular RNA was extracted and RT-PCR was used to measure the mRNA content of GPR31 gene (3 independent replicates each with 2 replicates), the results are shown in FIG. 9. The GPR31 knock-down group (shGPR31) had significantly reduced mRNA content of GPR31 gene compared to the shRNA control group.

The results of oil red O staining are shown in fig. 10, the control group had no distinct red color, while the cells stained with oil red O were significantly increased compared to the control group when PA + OA stimulation was added, and the GPR31 knock-down group (shGPR31 experimental group) had a smaller increase in the stained area than the shRNA experimental group. This result demonstrates that a decrease in GPR31 expression can inhibit PA-stimulated lipid deposition in L02 cells.

Example 6 Effect of GPR31 overexpression on myocardial hypertrophy

H9C2 cells were divided into 4 groups: GFP control group, GPR31 control group, GFP Ang II group, GPR31 Ang II group. The corresponding lentiviral virus solutions infected cultured H9C2 cells, respectively, 24H later, stimulated with 1 μ M angiotensin II (Ang II) or PBS (control) for 48H, and then subjected to immunofluorescence assay. After lentivirus transfection is completed, extracting total cell protein, and detecting the expression condition of GPR31 protein in H9C2 cells by Western blot. After the completion of Ang II stimulation, cells were harvested for RT-PCR analysis (2 independent replicates, 3 technical replicates each time) to detect the mRNA content of the hypertrophy marker gene Anp and Myh 7. Cell surface area statistics were calculated as GFP control value of 1 and the ratio of the remaining groups to the group was calculated.

The primer sequences used for RT-RCR are as follows:

gene	Forward primer	Reverse primer
			Anp	ACCTGCTAGACCACCTGGAG	CCTTGGCTGTTATCTTCGGTACCGG
Myh7	CCGAGTCCCAGGTCAACAA	CTTCACGGGCACCCTTGGA

The results of detecting the expression of GPR31 protein in H9C2 cells were the same as in example 3.

The results of the H9C2 cell hypertrophy and myocardial hypertrophy marker expression tests are shown in fig. 11, when Ang ii treatment is performed, the cell surface area is significantly increased compared to the PBS control group, and the cell enlargement degree of GPR31 overexpression group is significantly greater than that of the GFP Ang ii group (fig. 11A, B); consistent with the above results, mRNA analysis showed that the degree of upregulation of GPR31 overexpression cellular mast marker genes Anp and Myh7 was significantly higher in the cells treated with Ang ii than in the control group (fig. 11C).

Claims

Use of a GPR31 inhibitor for the manufacture of a medicament for the treatment of cardiac ischemia reperfusion injury;

the inhibitor is shRNA of mRNA of GPR31, and the interference targeting sequence of the inhibitor is CACTCTCCTGCCTTCAGTTTG.
2. The use of claim 1, wherein the shRNA sequence is: the forward oligonucleotide: 5'-CCGGCACTCTCCTGCCTTCAGTTTGCTCGAGCAAACTGAAGGCAGGAGAGTGTTTTTG-3' and the reverse oligonucleotide: 5'-AATTCAAAAACACTCTCCTGCCTTCAGTTTG CTCGAGCAAACTGAAGGCAGGAGAGTG-3'.
3. The use of claim 1 or 2, wherein the triggering factor for the ischemic reperfusion injury of the heart is selected from the group consisting of myocardial infarction, myocardial infarction recanalization injury, heart transplantation, coronary thrombolysis, percutaneous coronary angioplasty, intracoronary dilatation and/or coronary bypass.
4. The use of claim 1 or 2, the medicament further comprising a pharmaceutically acceptable excipient.
5. Use of a vector expressing an shRNA targeting the mRNA of GPR31 in the manufacture of a medicament for the treatment of cardiac ischemia reperfusion injury;

the shRNA interference targeting sequence is CACTCTCCTGCCTTCAGTTTG.
6. The use of claim 5, wherein the shRNA sequence is: the forward oligonucleotide: 5'-CCGGCACTCTCCTGCCTTCAGTTTGCTCGAGCAAACTGAAGGCAGGAGAGTGTTTTTG-3' and the reverse oligonucleotide: 5'-AATTCAAAAACACTCTCCTGCCTTCAGTTTG CTCGAGCAAACTGAAGGCAGGAGAGTG-3'.
7. The use of claim 5 or 6, wherein the triggering factor for the ischemic reperfusion injury in the heart is selected from the group consisting of myocardial infarction, myocardial infarction recanalization injury, heart transplantation, coronary thrombolysis, percutaneous coronary angioplasty, intracoronary dilatation, and/or coronary bypass.
8. The use according to claim 5 or 6, wherein the vector is an expression vector comprising a promoter and a transcription termination sequence operably linked to the shRNA sequence.
9. The use of claim 8, wherein the expression vector is a eukaryotic cell expression vector.
10. The use of claim 9, wherein the eukaryotic cell expression vector is a plasmid expression vector or a viral expression vector.
11. The use of claim 10, wherein the plasmid expression vector is selected from the group consisting of pcDNA3.1+/-, pcDNA4/HisMax B, pSecTag 2A, pVAX1, pBudCE4.1, pTracer CMV2, pcDNA3.1(-)/Myc-His A, pcDNA6-Myc/His B, pCEP4, pIRES, pIRESneo, pIRES hyg3, pCMV-Myc, pCMV-HA, RESpI-puro 3, pIRES-neo3, pCAGGS, pSilencer1.0, pSilencer2.1-U6 hygro, pSilencer3.1-H1 hygro, pSilencer3.1-H1 neo, and pSilencer4.1-CMV neo.
12. The use of claim 10, wherein the viral expression vector is selected from the group consisting of a lentiviral vector, an adenoviral vector, and an adeno-associated viral expression vector.
13. The use of claim 12, wherein the viral expression vector is selected from the group consisting of plko.1, pLVX-IRES-ZsGreen1, pCDH-EF1-Luc2-T2A-tdTomato, pCDH-MSCV-MCS-EF1-Puro, pCDH-MSCV-MCS-EF1-copGFP, pLVX-ZsGreen1-C1, pAdEasy-1, pShuttle-CMV, pShuttle, pAdTrack-CMV, pShuttle-IRES-hrGFP-1, pShuttle-IRES-hrGFP-2, pShuttle-CMV-lacZ, pShuttle-CMV-EGFP-xc 1, pBHGE3, pamcs-hgs, parc, pelperv, or pAAV-lacZ.
14. Use of a lentiviral vector comprising an shRNA targeting an mRNA of GPR31 in the manufacture of a medicament for the treatment of cardiac ischemia reperfusion injury;

the shRNA interference targeting sequence is CACTCTCCTGCCTTCAGTTTG.
15. The use of claim 14, wherein the shRNA sequence is: 5 'CCGGCACTCTCCTGCCTTCAGTTTGCTCGAGCAAACTGAAGGCAGGAGAGTGTTTTTG 3' for the forward oligonucleotide and 5 'AATTCAAAAACACTCTCCTGCCTTCAGTTTG CTCGAGCAAACTGAAGGCAGGAGAGTG 3' for the reverse oligonucleotide.
16. The use of claim 14 or 15, wherein the triggering factor for the ischemic reperfusion injury in the heart is selected from the group consisting of myocardial infarction, myocardial infarction recanalization injury, heart transplantation, coronary thrombolysis, percutaneous coronary angioplasty, intracoronary dilatation, and/or coronary bypass.
17. The use of claim 14 or 15, wherein the lentiviral vector is a plko.1 vector.
18. The use according to any one of claims 5 or 6 or 14 or 15, wherein the medicament further comprises a pharmaceutically acceptable carrier.
19. The use of claim 18, wherein the pharmaceutical carrier is an injection carrier.
20. The use of claim 19, wherein the injection carrier is selected from the group consisting of: isotonic NaCl solution, isotonic glucose solution, isotonic solution containing buffer system.