阅读说明：本技术 Ctrp13脂肪因子的新用途 (New application of CTRP13 fat factor ) 是由 王成 于 2021-08-02 设计创作，主要内容包括：本发明属于医药领域,具体公开了CTRP13脂肪因子的新用途。CTRP13在制备PDGFRβ磷酸化抑制剂和/或PDGFR下游信号通路抑制剂中的应用。PDGFRβ磷酸化和/或PDGFR下游信号通路的抑制剂,包括CTRP13。PDGFRβ磷酸化和/或PDGFR下游信号通路抑制剂在制备平滑肌表型转换抑制药剂中的应用。CTRP13在制备平滑肌表型转换抑制药剂中的应用。本发明提供CTRP13对PDGFRβ有抑制作用,因此CTRP13可以抑制平滑肌表型转换,从而预防和/或治疗血管损伤后再狭窄；此外,CTRP13并无通常心血管药物或其他平滑肌表型转换抑制剂的细胞毒作用,因此可以预期其安全性；其可以作为治疗平滑肌参与的相关疾病如血管再狭窄、肿瘤等疾病的药物而广泛应用。(The invention belongs to the field of medicines, and particularly discloses a new application of a CTRP13 fat factor. Application of CTRP13 in preparing PDGFR beta phosphorylation inhibitor and/or PDGFR downstream signal pathway inhibitor. Inhibitors of PDGFR β phosphorylation and/or PDGFR downstream signaling pathways, including CTRP 13. Use of an inhibitor of PDGFR β phosphorylation and/or PDGFR downstream signaling pathway in the preparation of a medicament for inhibiting smooth muscle phenotype switching. Use of CTRP13 in the manufacture of a smooth muscle phenotype switch inhibitory medicament. The invention provides that CTRP13 has an inhibitory effect on PDGFR beta, so that CTRP13 can inhibit smooth muscle phenotype switching, thereby preventing and/or treating restenosis after vascular injury; furthermore, CTRP13 has no cytotoxic effect of common cardiovascular drugs or other inhibitors of smooth muscle phenotype switching, and therefore its safety can be expected; it can be widely used as a medicine for treating related diseases involving smooth muscle, such as vascular restenosis, tumor and the like.)

Application of CTRP13 fat factor in preparing PDGFR beta inhibitor.

Application of the CTRP13 fat factor in preparing PDGFR beta phosphorylation inhibitor and/or PDGFR beta downstream signal pathway inhibitor.

An inhibitor of PDGFR β phosphorylation and/or PDGFR β downstream signaling pathways comprising CTRP 13.

Use of an inhibitor of PDGFR β phosphorylation and/or PDGFR β downstream signaling pathway in the preparation of a smooth muscle phenotype switch inhibitory medicament.

Use of CTRP13 adipokine in the manufacture of a medicament for inhibiting smooth muscle cell phenotypic transition.

6. An agent for inhibiting smooth muscle cell phenotypic transition, comprising at least one of:

a.CTRP13，

an inhibitor of phosphorylation of PDGFR-beta,

a pdgfr β downstream signaling pathway inhibitor.

Application of the CTRP13 fat factor in preparing medicines for preventing and treating vascular diseases.

The application of the CTRP13 fat factor in preparing the medicine for preventing and treating the vascular restenosis.

9. A medicament for the prevention or treatment of vascular disease or restenosis, comprising at least one of:

a factor of the fat content of CTRP13,

an inhibitor of phosphorylation of PDGFR-beta,

a PDGFR beta downstream signaling pathway inhibitor,

d. smooth muscle phenotype transition inhibitory agents.

10. The medicine for preventing and treating the abdominal aortic aneurysm disease caused by hypertension is characterized by comprising CTRP13 fat factor.

Technical Field

The invention belongs to the field of medicines, and particularly relates to novel applications of a CTRP13 fat factor.

Background

Human C1q tumor necrosis factor-related protein 13, british name CTRP13, alias complete C1q Like 3; c1q And turbine promoter-Related Protein 13; compact Component 1, Q Subcomponent-Like 3; compact C1q-Like Protein 3; contains 255 amino acids and has a molecular weight of 26719 Da.

NCBI reference sequence No. NP-001010908.1, amino acid sequence as follows:

MVLLLVILIPVLVSSAGTSAHYEMLGTCRMVCDPYGGTKAPSTAATPDRGLMQSLPTFIQGPKGEAGRPGKAGPRGPPGEPGPPGPMGPPGEKGEPGRQGLPGPPGAPGLNAAGAISAATYSTVPKIAFYAGLKRQHEGYEVLKFDDVVTNLGNHYDPTTGKFTCSIPGIYFFTYHVLMRGGDGTSMWADLCKNNQVRASAIAQDADQNYDYASNSVVLHLEPGDEVYIKLDGGKAHGGNNNKYSTFSGFIIYAD。

in recent years, the study of members of the complement C1q/TNF-related proteins (C1q/TNF-related proteins, CTRPs) family for metabolic disorders has attracted considerable attention, including metabolic syndrome and diabetes. The CTRP13 fat factor (hereinafter referred to as CTRP13) is a highly conserved novel fat factor in CTRPs family members, and is involved in the regulation and control of cardiovascular and metabolic related diseases. Studies have shown that blood glucose levels affect the expression of CTRP13, and multiple lines of evidence from different populations have demonstrated that serum CTRP13 expression levels are significantly reduced in metabolic diseases including fatty liver, diabetes. Consistent with these studies, our previous studies also found that CTRP13 was able to significantly inhibit foam cell formation, delaying the development of atherosclerosis. Meanwhile, the CTRP13 was also found to reduce the incidence of renal failure vascular calcification and hypertension-induced abdominal aortic aneurysm, so we speculate that CTRP13 may also play an important role in vascular biology, particularly in the regulation of the development of vascular remodeling. It is not clear whether CTRP13 has potential as a drug for restenosis following vascular injury.

Vascular restenosis is the re-narrowing of the lumen of an artery following treatment by interventional procedures (including vascular surgery, cardiac surgery, angioplasty, and the like), and is typically measured 3-8 months after revascularization, and is arbitrarily defined as a permanent reduction in vessel diameter of greater than 50% compared to a reference artery. When a patient is subjected to coronary intervention (PCI) operation, endothelial cells are damaged and stripped by implanting the stent, and blood vessels respond to damage stimulation, so that a series of reactions such as inflammatory reaction, thrombosis, fibrin deposition, platelet aggregation and leukocyte recruitment are induced, and finally extracellular matrix deposition and SMCs proliferation are caused. On the one hand, stent implantation greatly improves clinical outcomes. On the other hand, it will cause a vascular injury response, coupled with the action of a chronic indwelling stent, the inflammatory cascade being a key factor in the initiation of restenosis.

The phenotypes of Vascular Smooth Muscle Cells (VSMCs) can be classified into a more differentiated contractile phenotype and a less differentiated secretory phenotype, which represent two extreme types of a series of different phenotypes coexisting in the vessel wall and expressing different genes and proteins. The VSMC of normal adult arterial blood vessels predominates in the systolic form, whose main functions are to maintain the elasticity of the blood vessels and to constrict the blood vessels. The contractile VSMC has poor or no proliferation and migration capacity, and the soma is fusiform or banded and contains a large amount of myofilaments and structural proteins; secreted VSMCs, on the other hand, are mainly present in the metaphase and pathological vessels of the embryo, and their main functions are proliferation, migration into the intima, and synthesis of extracellular matrix proteins. The process of VSMC switching from systolic to secretory is called phenotypic switching of VSMC. Research shows that three signal transduction pathways, namely mitogen-activated protein kinase (MAPK), PI-3-K and cyclic adenosine monophosphate (cAMP), are involved in phenotypic transformation of VSMC through receptors such as VEGFR (VEGFR) and platelet-derived growth factor receptor (PDGFR). Abnormal proliferation and migration of VSMC are common pathological features of occurrence and development of vascular diseases such as hypertension, pulmonary hypertension and the like, and are also important reasons for restenosis after vascular injury, and VSMC phenotypic transformation plays an important role in VSMC proliferation and migration.

Many drugs are currently available for the prevention of restenosis, including antiplatelet and anticoagulant drugs, statins, angiotensin converting enzyme inhibitors, vitamins and some antiproliferative drugs. But there is increasing evidence that these drugs have less pronounced prophylactic effects on restenosis. Currently, the main drugs with good clinical effect and low side effect in the local application of drugs are antiproliferative drugs, such as sirolimus and its derivatives (such as everolimus, zotarolimus, etc.) and paclitaxel, which have been widely used in drug-coated stents. Pioglitazone, tranilast, anti-inflammatory drugs such as prednisone, colchicine and the like are currently in clinical trials, and are expected to be applied to clinical treatment in the future. The existing research shows that the CTRP13 has the functions of regulating cell signal molecules and transcriptional coordination activity, and the role of the CTRP13 in restenosis after vascular injury is not researched.

Disclosure of Invention

Aiming at the problems, the invention provides novel applications of CTRP13 and novel therapeutic drugs for diseases, and mainly solves the problems of the existing CTRP13 and fills up the blank of research in certain vascular diseases.

In order to solve the problems, the invention adopts the following technical scheme:

application of CTRP13 fat factor in preparing PDGFR beta inhibitor.

Application of CTRP13 in preparing PDGFR beta phosphorylation inhibitor and/or PDGFR downstream signal pathway inhibitor.

Inhibitors of PDGFR β phosphorylation and/or PDGFR downstream signaling pathways, including CTRP 13.

Use of an inhibitor of PDGFR β phosphorylation and/or PDGFR downstream signaling pathway in the preparation of a medicament for inhibiting smooth muscle phenotype switching.

Use of CTRP13 in the manufacture of a smooth muscle phenotype switch inhibitory medicament.

An agent for inhibiting phenotypic transition of smooth muscle cells or vascular smooth muscle cells, comprising at least one of:

a.CTRP13，

an inhibitor of phosphorylation of PDGFR-beta,

a pdgfr downstream signaling pathway inhibitor.

Application of CTRP13 in preparing medicine for preventing and treating vascular diseases.

Application of CTRP13 in preparing medicine for preventing and treating vascular restenosis is provided.

The medicine for preventing and treating vascular diseases at least comprises one of the following medicines:

a.CTRP13，

an inhibitor of phosphorylation of PDGFR-beta,

an inhibitor of the downstream signalling pathway of PDGFR,

d. smooth muscle phenotype transition inhibitory agents.

In some embodiments, the vascular disease is restenosis.

Application of CTRP13 in preparing foam cell inhibitor.

Foam cell inhibitors including CTRP 13.

Application of CTRP13 in preparing medicine for preventing and treating renal failure vascular calcification and/or abdominal aortic aneurysm disease caused by hypertension.

A medicine for preventing and treating the renal failure, vascular calcification and/or abdominal aortic aneurysm caused by hypertension contains CTRP 13.

The invention has the beneficial effects that:

CTRP13 has inhibitory effects on PDGFR β, and therefore CTRP13 inhibits smooth muscle phenotype switching, thereby preventing and/or treating restenosis following vascular injury. Furthermore, CTRP13 has no cytotoxic effect of common cardiovascular drugs or other inhibitors of smooth muscle phenotype switching, and therefore its safety can be expected. It can be widely used as a medicine for treating related diseases involving smooth muscle, such as vascular restenosis, tumor and the like.

Drawings

FIG. 1 shows that a is a group of a pseudo-operation and a carrier treatment, b is a group of a ligation and a carrier treatment, c is a group of a ligation and a CTRP13 treatment, and then the blood vessel morphology of a rat after carotid artery sacculus injury is detected by an oil red-Hematoxylin (HE) staining experiment;

FIG. 2 shows VSMC proliferation assay using EdU assay with different concentrations of CTRP13 and vehicle (DMSO);

FIG. 3 shows the expression of cyclin and oncostatin measured by Western blot analysis of VSMC cells treated with PDGF-BB followed by CTRP13(100,200,300ng/ml) and vehicle (DMSO) stimulation, respectively;

FIG. 4 shows that after VSMC is treated with PDGF-BB, CTRP13(100,200,300ng/ml) and carrier (DMSO) are respectively given for stimulation, and the expression of apoptosis-related proteins is detected by Western blotting with CTRP13 and carrier at different concentrations;

FIG. 5 shows the migration of cells measured by transwell assay with different concentrations of CTRP13 following PDGF-BB treatment of VSMC cells, stimulated by CTRP13(100,200,300ng/ml) and vehicle (DMSO), respectively;

FIG. 6 shows that after PDGF-BB treatment of SMC cells, CTRP13(100,200,300ng/ml) and vehicle (DMSO) stimulation were respectively given, and CTRP13 at different concentrations was used to detect cell proliferation migration by using a scratch test;

FIG. 7 shows the measurement of the expression of the contractile gene α SMA by immunofluorescence assay of CTRP13 at various concentrations after the injury of the carotid artery sacculus of a rat, which was treated with vehicle and 10mg/kg dCTRP13, respectively;

FIG. 8 shows PDGFR β phosphorylation in VSMC treated with PDGF-BB followed by CTRP13(100,200,300ng/ml) and vehicle (DMSO) stimulation at different concentrations of CTRP13 using Western blotting.

Detailed Description

The invention is further illustrated below:

application of CTRP13 in preparing PDGFR beta phosphorylation inhibitor and/or PDGFR downstream signal pathway inhibitor.

Inhibitors of PDGFR β phosphorylation and/or PDGFR downstream signaling pathways, including CTRP13 fat factor.

Use of an inhibitor of PDGFR β phosphorylation and/or PDGFR downstream signaling pathway in the preparation of a medicament for inhibiting smooth muscle phenotype switching.

Use of CTRP13 adipokine in the manufacture of a medicament for inhibiting smooth muscle phenotype transition.

The smooth muscle phenotype switch is mainly expressed as a smooth muscle cell phenotype switch.

A smooth muscle phenotype switching inhibitory agent comprising at least one of:

a factor of the fat content of CTRP13,

an inhibitor of phosphorylation of PDGFR-beta,

a pdgfr downstream signaling pathway inhibitor.

Application of CTRP13 in preparing medicine for preventing and treating vascular diseases.

Application of CTRP13 in preparing medicine for preventing and treating vascular restenosis is provided.

The medicine for preventing and treating vascular diseases at least comprises one of the following medicines:

a factor of the fat content of CTRP13,

an inhibitor of phosphorylation of PDGFR-beta,

an inhibitor of the downstream signalling pathway of PDGFR,

d. smooth muscle phenotype transition inhibitory agents.

The vascular disease is restenosis of the blood vessel. Restenosis after vascular injury includes PCI restenosis, in-stent restenosis, and restenosis after bypass grafting.

Application of CTRP13 fat factor in preparing foam cell inhibitor.

Foam cell inhibitors including CTRP13 adipokine.

Application of the CTRP13 fat factor in preparing medicine for preventing and treating abdominal aortic aneurysm diseases caused by renal failure vascular calcification and/or hypertension.

A medicine for preventing and treating the renal failure, vascular calcification and/or abdominal aortic aneurysm caused by hypertension contains CTRP13 fat factor.

The following examples use cell experiments to study the inhibitory effect of the drug on VSMC phenotypic switching.

The CTRP13 fat factor used in the testing of this example was purchased from Aviscera Bioscience, model No. 00333-01-100. The following procedures were used to examine vascular lesion restenosis and VSMC phenotypic shift in the following experiments, respectively.

And detecting the VSMC cell proliferation condition by using an EdU cell proliferation experiment, and detecting the VSMC cell migration condition by using a cell scratch experiment and a transwell experiment. The detection method is described in Huang D, Wang Y, Wang L, Zhang F, Deng S, Wang R, Zhang Y, Huang K.Poly (ADP-rib) polymerase 1is Induced dependent for transforming growth factor-beta Induced Smad3 activation in vascular smooth cell. PLoS one.2011; e27123. the specific process is as follows:

EdU cell proliferation assay: rat-derived primary cells (VSMC) were seeded in 96-well plates and cells were treated with different concentrations (100,200,300ng/ml) of CTRP13 and vehicle DMSO, respectively, for 4 h. After 48h, the four treatments were stimulated with PDGF-BB (30ng/ml) for 48h (control with an equal volume of DSMO), and EdU incorporation was analyzed according to the manufacturer's instructions and photographed with an Olympus cellSens Entry.

Cell scratch test: VSMC were seeded into 6-well plates and cultured to 80% density. The cell monolayer was scratched with a 1ml pipette tip. After pre-incubation of cells with different concentrations of CTRP13 for 4h, cells were stimulated with PDGF-BB (30ng/ml) for 48h (control with equal volume of DSMO) and then cultured in DMEM containing fetal bovine serum at 10% volume concentration. Cells were visualized using an Olympus cellSens entry and wound closure rate was measured using the Image J program.

Cell migration was measured by the Transwell method: VSMC was pretreated with CTRP13 for 4h, seeded in the upper air cell, 500L DMEM and fetal bovine serum at 10% concentration by volume and PDGF-BB (30ng/ml) was placed in the lower air cell. After 24h, the cells were fixed in the lower air chamber with 4% by mass of formaldehyde for 20 minutes and stained with 0.1% by mass of crystal violet for 20 minutes. Migrated cells were photographed using the Olympus cellSens channel.

Example 1

After injury of carotid sacculus or sham-operated injury of SD rats, CTRP13(10 mg/kg. d) and vehicle (DMSO) were intraperitoneally injected, respectively. After 14 days, the mice were euthanized and the injured blood vessels were subjected to a vascular resection procedure. After paraffin is fixed and embedded by formaldehyde with the mass concentration of 4%, the blood vessel is cut into sections. Fig. 1 shows the blood vessel morphology of a rat carotid artery after balloon injury is detected by an oil red-Hematoxylin (HE) staining experiment after the treatment of a sham operation + carrier treatment group, a balloon + carrier treatment group and a balloon + CTRP13 treatment group. The results are shown in FIG. 1. Wherein, the fig. 1is a rat carotid artery average thickness statistical chart from left to right respectively for a sham operation + carrier treatment group, a balloon + carrier treatment group and a balloon + CTRP13 treatment group. As can be seen from fig. 1, when rats underwent carotid balloon injury, carotid artery thickness increased, i.e., vascular injury resulted in vascular endothelial cell hyperproliferation, compared to the sham surgery + vehicle treatment group; and the CTRP13 can inhibit the excessive proliferation of vascular endothelial cells caused by vascular injury.

Example 2

VSMC cells were treated with PDGF-BB (30ng/ml) and then stimulated with different concentrations of CTRP13(100,200,300ng/ml) and vehicle DMSO, respectively. FIG. 2 shows the detection of VSMC proliferation using EdU assay at various concentrations of CTRP 13. The first column is the vector treated group, the second is the PDGF-BB and vector treated group, the third is the PDGF-BB and the CTRP13 treated group of 100ng/ml, the fourth is the PDGF-BB and the CTRP13 treated group of 200ng/ml, and the fifth is the PDGF-BB and the CTRP13 treated group of 300 ng/ml. As can be seen from fig. 2, after VSMC cells were treated with PDGF-BB, EdU-positive cells increased, and after further treatment with CTRP13, EdU-positive cells decreased, and the amount of EdU-positive cells decreased as the concentration of CTRP13 increased. In conclusion, after the VSMC cells are treated by PDGF-BB, the proliferation of the VSMC cells can be promoted, and the PDGF-BB-induced cell proliferation can be inhibited by CTRP13, and the inhibition effect is enhanced along with the increase of the concentration of CTRP 13.

Example 3

After VSMC cells were treated with PDGF-BB (30ng/ml), they were stimulated with CTRP13(100,200,300ng/ml) and DMSO (carrier) at different concentrations, and after collecting the cells and extracting the proteins, the expression of cyclin and oncostatin was examined by Western blotting, as shown in FIG. 3. VSMC cells expressed cyclin (PCNA, CyclinD1) and oncostatin (P27, P21) under different treatments. As shown in FIG. 3, after treatment with PDGF-BB, the expression of cyclin was increased and the expression of oncostatin was decreased in VSMC cells; after the stimulation of CTRP13, the expression of cyclin is reduced and the expression of cancer suppressor protein is increased; the expression level of the protein related to the apoptosis has no obvious change.

Example 4

VSMC cells were treated with PDGF-BB (30ng/ml), stimulated with CTRP13(100,200,300ng/ml) and DMSO (carrier) at different concentrations, collected, and protein was extracted and assayed for apoptotic protein expression (Caspase 3, cleaved Caspase 3, Bcl-2, Bax) by Western blotting, as shown in FIG. 4. As can be seen from FIG. 4, the expression of apoptosis-related proteins was not significantly affected by the concentration of CTRP 13.

Example 5

VSMC cells were treated with PDGF-BB (30ng/ml) and stimulated with different concentrations of CTRP13(100,200,300ng/ml) and vehicle DMSO, respectively, to detect cell migration using the transwell assay. The results are shown in FIG. 5. As can be seen from FIG. 5, PDGF-BB promotes cell migration, whereas CTRP13 inhibits cell migration, and the inhibitory effect increases with increasing concentration.

Example 6

After PDGF-BB treatment (30ng/ml) of VSMC cells, different concentrations of CTRP13(100,200,300ng/ml) and DMSO (carrier) were administered for stimulation, and cell proliferation and migration were examined by scratch assay, and the results are shown in FIG. 6. As can be seen from fig. 6, PDGF-BB promoted the closure rate of cell scratch, while CTRP13 inhibited the promotion, and the inhibition of cell scratch closure was increased with the increase in CTRP13 concentration.

Example 7

After the rat carotid sacculus is injured, a rat carrier DMSO and 10 mg/kg. d CTRP13 are respectively given to treat the carotid sacculus, the expression condition of the contractile gene alpha SMA is detected by an immunofluorescence experiment, and a tissue section is incubated overnight by using SM alpha-actin primary antibody (the volume ratio is 1: 100)4oC, and then incubated for 2 hours by using FITC-conjugated fluorescent secondary antibody 37 oC. Nucleic acids were stained with DAPI 37oC for 15 min. The sections were finally visualized using an Olympus cellSens entry. Carotid morphology was observed and the results are shown in FIG. 7. In fig. 7, the first column is the sham-surgery plus empty treatment group, the second column is the surgery plus empty treatment group, and the third column is the surgery plus CTRP13 treatment group. As shown in FIG. 7, after the rat underwent carotid balloon injury, the carotid wall was thickened, and the expression level of the contractile gene α SMA in the carotid was decreased; after further treatment with CTRP13, the carotid wall thickness decreased and the expression level of contractile gene α SMA increased. After the blood vessel is damaged, the blood vessel wall is thickened, and the expression quantity of the contraction gene alpha SMA is reduced.

Example 8

After VSMC cells were treated with PDGF-BB, they were stimulated with different concentrations of CTRP13(100,200,300ng/ml) and vehicle DMSO, and the PDGFR β phosphorylation was detected by Western blotting, as shown in FIG. 8. FIG. 8 shows that PDGFR β phosphorylation was increased after treatment of VSMC with PDGF-BB; PDGFR β phosphorylation levels decreased after further treatment with CTRP13, and decreased with increasing CTRP13 concentration. Thus, PDGF-BB promotes phosphorylation of PDGFR β, while CTRP13 inhibits phosphorylation of PDGFR β, and this inhibition increases with increasing concentration of CTRP 13.

CTRP13 was able to directly inhibit restenosis following vascular injury and phenotypic switching of VSMCs.

It will be apparent to those skilled in the art that various modifications may be made to the above embodiments without departing from the general spirit and concept of the invention. All falling within the scope of protection of the present invention. The protection scheme of the invention is subject to the appended claims.