Use of resistin in the preparation of a medicament for heart disease

Abstract

The application relates to the field of biological medicine, and discloses application of resistin in preparation of heart disease drugs. The application finds that resistin has the functions of inhibiting heart fibrosis and promoting cardiovascular angiogenesis, thereby having the potential of preparing heart disease (cardiovascular disease) drugs, and is expected to provide a new theoretical basis and a potential intervention target for heart failure.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, and particularly relates to application of resistin in preparation of a drug for heart disease. BACKGROUND

[0002] Tissue remodeling after heart injury is considered as the main pathological mechanism leading to the decline of heart function. The performance of heart tissue remodeling includes activation of renin-angiotensin-aldosterone system, fibrosis and decrease of blood vessel density. The existing treatment measures in clinic include drugs which mainly reduce the load of heart to delay the heart remodeling by inhibiting renin-angiotensin-aldosterone system. How to regulate the fibrosis process after heart injury and promote the angiogenesis of heart is an urgent problem to be solved in the field of cardiovascular disease research.

[0003] Heart disease includes myocardial infarction, pathological hypertrophy and heart failure which are often manifested as sterile injury. Sterile injury can lead to a series of inflammatory reactions. Tissue-resident macrophages play the role of "immune surveillance", and the ultimate goal is to restore the damaged tissue to homeostasis. The heart tissue is rich in immune cells, and the number of immune cells contained therein is more than 12 times that of skeletal muscle. Most of the immune cells are macrophages, which are considered to be a relatively uniform cell group and highly express RELMα.

[0004] RELMα belongs to the resistin family, and human resistin is extremely similar to mouse RELMα in origin and function, that is, they are mainly secreted by mononuclear macrophages and have the functions of regulating metabolism and immunity. Previous studies suggest that RELMα plays an important role in metabolic homeostasis and inflammation. Currently, there is no literature report on the role of resistin in protecting tissue remodeling after heart injury, especially the role of RELMα in inhibiting cardiac fibrosis and promoting angiogenesis. SUMMARY

[0005] In order to solve the above technical problems, the present application provides application of resistin in preparation of a drug for heart disease. The present application first discovers that resistin has the functions of inhibiting cardiac fibrosis and promoting angiogenesis, thereby having the potential to prepare a drug for heart disease (cardiovascular disease), and is expected to provide a new theoretical basis and potential intervention target for heart failure.

[0006] The specific technical scheme of the present application is as follows:

[0007] In the first aspect, the present application provides application of resistin in preparation of a drug for heart disease.

[0008] As described in the background section of the present application, the role of resistin in regulating metabolism and inflammation in many species including humans is relatively clear in the prior art, and there is currently no international report on the role of resistin in the heart, especially in heart failure. The present application first proposes that resistin can inhibit fibrosis and promote angiogenesis, which is expected to provide a new theoretical basis and potential intervention target for heart failure. Specifically, the present application finds that resistin has the ability to reduce fibrosis and promote angiogenesis by giving resistin treatment to a heart failure model, thereby having the potential to prepare a heart disease (cardiovascular disease) drug.

[0009] Preferably, the heart disease is heart failure.

[0010] Preferably, the heart disease drug comprises resistin as an active ingredient, and a pharmaceutically acceptable solvent, an excipient and / or a carrier.

[0011] Further preferably, the heart disease drug is an injection preparation and an oral preparation.

[0012] Further preferably, the injection preparation is an injection preparation by intramyocardial injection, subcutaneous injection, intramuscular injection or intravenous injection.

[0013] In a second aspect, the present application provides an application of resistin in the preparation of an anti-fibrosis drug.

[0014] Preferably, the anti-fibrosis drug is a drug for treating fibrosis diseases or a tool drug for scientific research.

[0015] Preferably, the fibrosis is cardiac fibrosis.

[0016] Preferably, the anti-fibrosis drug comprises resistin as an active ingredient, and a pharmaceutically acceptable solvent, an excipient and / or a carrier.

[0017] Preferably, the anti-fibrosis drug is an injection preparation and an oral preparation.

[0018] Further preferably, the injection preparation is an injection preparation by intramyocardial injection, subcutaneous injection, intramuscular injection or intravenous injection.

[0019] In a third aspect, the present application provides a method for inhibiting fibrosis of cardiac fibroblasts in vitro: by adding resistin to inhibit the expression level of Collagen protein, Periostin protein and a-SMA protein of cardiac fibroblasts under in vitro culture conditions, thereby inhibiting fibrosis of cardiac fibroblasts.

[0020] The present application finds, through in-vitro tests, that resistin can significantly reduce the expression level of fibrosis markers of cardiac fibroblasts, thus indicating its function of inhibiting fibrosis of cardiac fibroblasts.

[0021] In a fourth aspect, the present application provides use of resistin in preparation of a blood vessel neogenesis promoter.

[0022] Preferably, the blood vessel neogenesis promoter is a drug for treating diseases or a tool drug for scientific research.

[0023] Preferably, the blood vessel is a cardiac blood vessel.

[0023] Preferably, the blood vessel neogenesis promoter comprises resistin as an active ingredient, and a pharmaceutically acceptable solvent, an excipient, and / or a carrier.

[0024] Preferably, the blood vessel neogenesis promoter is an injection preparation and an oral preparation.

[0025] Further preferably, the injection preparation is an injection preparation through intramyocardial injection, intradermal injection, subcutaneous injection, intramuscular injection, or intravenous injection.

[0026] In a fifth aspect, the present application provides a method for promoting tube formation of venous endothelial cells into neovasculature under in-vitro conditions: adding resistin to promote tube formation of venous endothelial cells into neovasculature under in-vitro culture conditions.

[0027] The present application finds, through in-vitro tests, that resistin can significantly increase the number of tube formation of venous endothelial cells, thus indicating its function of promoting blood vessel neogenesis.

[0028] Compared with the prior art, the present application has the following beneficial effects: the present application first finds that resistin has the functions of inhibiting cardiac fibrosis and promoting cardiovascular neogenesis, thus having the potential to prepare a drug for cardiac diseases (cardiovascular diseases), and is expected to provide a new theoretical basis and a potential intervention target for heart failure.

[0029] BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1 Figure 9 is a comparison chart of ultrasonic Doppler evaluation of cardiac function indicators of mice after 9 weeks of post-operation of the sham group (sham group), the control group (TAC), and the resistin (RELMa) administration group (TAC+RELMa) of the mouse aortic constriction model; wherein: A is the left ventricular interventricular septum thickness (LVID;d) during diastole; B is the left ventricular ejection fraction (LVEF); C is the left ventricular fractional shortening (FS); *P<0.05; **P<0.01; ***P<0.001.

[0031] Figure 2Aortic constriction model was performed on mice, and the heart tissue was taken after 9 weeks of postoperative sham operation group (sham group), control group (TAC), resistin (RELMa) administration group (TAC+RELMa) for fibrosis and angiogenesis detection. A is the picture of fibrosis Masson staining; scale: 300 um; B is the quantitative result of evaluating cardiac fibrosis; C. is the CD31 staining picture; scale: 100 um; D is the quantitative result of heart blood vessels; * P<0.05; **P<0.01; ***P<0.001.

[0032] Figure 3 The rat fibroblasts were cultured in vitro, and different concentrations of RELMα (0, 1, 2, 4, 8 ug / ml) were added to the culture. The comparison chart of each index after culture; among them: A is the Western blot detection of fibrosis indexes Collagen I, Periostin and a-SMA band; B is the quantitative result of Collagen I; C is the quantitative result of Periostin; D is the quantitative result of a-SMA. * P<0.05; **P<0.01; ***P<0.001.

[0033] Figure 4 Human umbilical vein endothelial cells (HUVEC cells) were cultured in vitro, and different concentrations of RELMα (0, 1, 2, 4, 8 ug / ml) were added after culture. Among them: A is the image of capturing cells at different time points; B is the quantitative result of the number of endothelial cell tubes after 2.5 hours (h); C is the quantitative result of the number of endothelial cell tubes after 4h; D is the quantitative result of the number of endothelial cell tubes after 6h; E is the quantitative result of the number of endothelial cell tubes after 12h. Scale: 200 um; * P<0.05; **P<0.01; ***P<0.001. DETAILED DESCRIPTION

[0034] The application will be further described below in combination with examples.

[0035] Overall examples

[0036] In a first aspect, the application provides the use of resistin in the preparation of a heart disease drug.

[0037] In some preferred embodiments, the heart disease is heart failure.

[0038] In some preferred embodiments, the heart disease drug comprises: resistin as an active ingredient, and a pharmaceutically acceptable solvent, adjuvant and / or carrier.

[0039] In some preferred embodiments, the heart disease drug is an injection preparation and an oral preparation.

[0040] In some more preferred embodiments, the injection preparation is an injection preparation by intramyocardial injection, subcutaneous injection, intramuscular injection, or intravenous injection.

[0041] In a second aspect, the present application provides use of resistin in the manufacture of an anti-fibrosis drug.

[0042] In some preferred embodiments, the anti-fibrosis drug is a drug for treating a fibrotic disease or a tool drug for scientific research.

[0043] In some preferred embodiments, the fibrosis is cardiac fibrosis.

[0044] In some preferred embodiments, the anti-fibrosis drug comprises resistin as an active ingredient, and a pharmaceutically acceptable solvent, an excipient, and / or a carrier.

[0045] In some preferred embodiments, the anti-fibrosis drug is an injection preparation and an oral preparation.

[0046] In some more preferred embodiments, the injection preparation is an injection preparation by intramyocardial injection, intradermal injection, subcutaneous injection, intramuscular injection, or intravenous injection.

[0047] In a third aspect, the present application provides a method for inhibiting fibrosis of cardiac fibroblasts in vitro, comprising: adding resistin to inhibit expression levels of Collagen protein, Periostin protein, and a-SMA protein in cardiac fibroblasts under in vitro culture conditions, thereby inhibiting fibrosis of cardiac fibroblasts.

[0048] In a fourth aspect, the present application provides use of resistin in the manufacture of an angiogenesis promoter.

[0049] In some preferred embodiments, the angiogenesis promoter is a drug for treating a disease or a tool drug for scientific research.

[0050] In some preferred embodiments, the blood vessel is a cardiac blood vessel.

[0051] In some preferred embodiments, the angiogenesis promoter comprises resistin as an active ingredient, and a pharmaceutically acceptable solvent, an excipient, and / or a carrier.

[0052] In some preferred embodiments, the angiogenesis promoter is an injection preparation and an oral preparation.

[0053] In some more preferred embodiments, the injection preparation is an injection preparation by intramyocardial injection, subcutaneous injection, intramuscular injection, or intravenous injection.

[0054] In a fifth aspect, the present application provides a method for promoting the tube formation of venous endothelial cells into new blood vessels in vitro: adding resistin to promote the tube formation of venous endothelial cells into new blood vessels in vitro. Specific embodiments Embodiments

[0056] (1) Construction of mouse heart failure model: After isoflurane gas anesthesia, 8-10 week old mice (Shanghai Slake Biological Co., Ltd.) were separated from the skin and muscle, and the suprasternal notch approach was used. Cut part of the sternum along the midline of the sternum. Use a small animal chest support to expose the field of view. Find the aortic arch, use a 6-0 suture to pass around the aortic arch, place a 27 gauge pad needle across the aortic arch and tie a knot, then remove the pad needle. Suture the sternum and skin, and place it on a warming pad until the mouse wakes up. The same surgical procedure but without the knot operation is used for the sham group. Drug administration group: start injecting resistin RELMα (Peprotech Company Cat# 450-26; 10 mg / each) into the abdominal cavity 6 weeks after the operation, twice a week for 4 weeks. The control group was injected with the corresponding solvent (0.1% BSA in PBS solution).

[0057] (2) Preoperative and postoperative application of ultrasound Doppler to evaluate mouse heart function.

[0058] (3) Sample collection: After 4 weeks of resistin injection, the mice were sacrificed after CO2 anesthesia, and the heart was collected. The heart tissue was fixed, OCT embedded and frozen sectioned, and CD31 antibody (BD Company Cat# 550274) and Masson staining kit (Solarbio Company) were used to quantitatively evaluate the degree of fibrosis and neovascularization, and imageJ software was used for quantitative analysis.

[0059] (4) Figure 1 Comparison chart of various indicators of heart function in different groups of mice after aortic constriction model (sham group, control group (TAC), resistin (RELMα) administration group (TAC+ RELMα)); Wherein: A is the left ventricular interventricular septal thickness (LVID;d) during diastole; B is the left ventricular ejection fraction (LVEF); C is the left ventricular fractional shortening (FS); Figure 2 Fibrosis and angiogenesis detection after 9 weeks of postoperative heart tissue sampling. A is the fibrosis Masson staining picture; B is the quantitative result of evaluating heart fibrosis; C. is the CD31 staining picture; D is the quantitative result of heart blood vessels.

[0060] As Figure 1As shown: after 9 weeks post-operation, the control group's cardiac systolic function indicators LVEF (51.5%) and FS value (26.7%) have been significantly decreased, while the RELMα administration group's LVEF value and FS value maintained at about 64.0% and 34.5%, indicating that the cardiac systolic function has been significantly improved compared with the control group. Further, as shown, the blood vessel density staining quantification of the administration group (6.6%) has significantly increased compared with the control group (4.8%), while the fibrosis area of the RELMα group (2.1%) has significantly decreased compared with the control group (5.9%). The above results show that in the mouse heart failure model, supplementing the resistin RELMα can improve the cardiac function by inhibiting fibrosis and promoting angiogenesis. Figure 2

[0061] Example 2

[0062] (1) P0 neonatal rat fibroblasts were separated by using a neonatal rat heart dissociation kit (Miltenyi Company, No: 130-098-373). Subsequently, these fibroblasts were transferred to a 10 cm culture dish and cultured overnight in a low-sugar DMEM medium containing 10% serum.

[0063] (2) The next day, the fibroblasts were digested with a trypsin solution and inoculated on a 6-well plate, and cultured again overnight in a low-sugar DMEM medium containing 10% serum. On the third day, the culture medium was changed to a serum-free low-sugar medium for further culture.

[0064] (3) After 24 hours of incubation, the culture medium was changed to a culture medium to which tumor growth factor β (concentration of 10 ng / ml) and different concentrations of RELMα (0 ug / ml, 1 ug / ml, 2 ug / ml, 4 ug / ml, 8 ug / ml) had been added for further culture.

[0065] ​(4) After 24 hours of drug treatment, proteins were extracted using RIPA lysis buffer (Beyotime). Protein samples (50 ug) were separated using 10% or 15% SDS-PAGE gels and transferred to PVDF membranes (Miltenyi). After blocking with 5% BSA solution, the primary antibody was incubated at 4°C overnight; the next day, after PBST washing, the HRP secondary antibody was incubated at room temperature for 1 hour, and after PBST washing, the ECL reagent (Merck Millipore) was used for color development, and Amersham Image Quant 800 was used for imaging. ImageJ software was used for quantitative analysis. Primary antibody information: Anti-mPeriostin / OSF-2 Goat IgG (R&D Cat# AF2955); Recombinant Anti-Collagen I antibody (Abcom Cat#ab270993); Rabbit polyclonal to alpha smooth muscle Actin (Abcom Cat#ab5694); HRP Conjugated Anti-GAPDH Recombinant Antibody (Huabio Cat#ET1702-66). Secondary antibody information: HRP Conjugated Goat anti-Rabbit IgG Goat Polyclonal Antibody (Huabio Cat# HA1001) Donkey Anti-Goat IgG H&L (HRP) (Abcam Cat#ab6885).

[0066] (4) The results are shown in Table 2: Figure 3 Collagen protein in the 2-8 ug / ml RELMα group was significantly lower than that in the simple TGFβ model group; Periostin protein in the 2 ug / ml RELMα group was significantly lower than that in the simple TGFβ model group; a-SMA protein in the 4 ug / ml RELMα group was significantly lower than that in the simple TGFβ model group. The above results show that the addition of RELMα can inhibit the fibroblast fibrosis process.

[0067] Example 3

[0068] (1) Matrigel (Corning) was added to the 96-well plate at a volume of 50 ul / well after overnight melting. The 96-well plate coated with Matrigel was placed in the cell culture incubator and incubated at 37°C for 30-60 minutes.

[0069] (2) The well-grown human umbilical vein endothelial cells (HUVECs) are resuspended with the endothelial cell complete culture medium containing 1% serum after being digested by Accutase enzyme.

[0070] (3) The cells are resuspended by adding the culture medium containing different concentrations of RELMα (0 ug / ml, 1 ug / ml, 2 ug / ml, 4 ug / ml, 8 ug / ml); the cells are transferred into a 96-well plate at a density of 1.7×10 4 / 100ul for culture.

[0071] (4) The 96-well plate is placed in a cell culture box for culture. The cell images are captured by using a bright field inverted microscope at 2.5, 4, 6, 12 hours, respectively. The imageJ software is used for quantitative analysis of the number of blood vessel tubes.

[0072] (5) The results are shown in the following table: Figure 4 At 2.5 hours (h), the number of blood vessel tubes in the 4-8 ug / ml RELMα group is significantly increased compared with the control group; at 4 h, the number of blood vessel tubes in the 4 ug / ml RELMα group is significantly increased compared with the control group; at 6 h, the number of blood vessel tubes in the 4 ug / ml RELMα group is significantly increased compared with the control group; at 8 h, the number of blood vessel tubes in the 4 ug / ml RELMα group is significantly increased compared with the control group. The above results show that the addition of RELMα can inhibit the fibroblast fibrosis process.

[0073] According to the experimental results of the above embodiments 1 to 3, the beneficial effects of the present application are: the present application first discovers that resistin has the effects of inhibiting cardiac fibrosis and promoting cardiovascular angiogenesis, thereby having the potential for preparing a drug for heart disease (cardiovascular disease), and is expected to provide a new theoretical basis and potential intervention target for heart failure.

[0074] The raw materials and equipment used in the present application are conventional raw materials and equipment in the art unless otherwise specified; the methods used in the present application are conventional methods in the art unless otherwise specified.

[0075] The above is only a preferred embodiment of the present application, and does not limit the present application in any way. Any simple modification, change and equivalent transformation of the above embodiment according to the technical essence of the present application still belongs to the protection scope of the technical solution of the present application.

Claims

1. Use of resistin RELMα for the preparation of a medicament for the treatment of heart failure, characterized in that: The heart failure is heart failure caused by aortic constriction; the drug is an injection preparation.

2. Use according to claim 1, characterized in that: The drug is a drug for treating heart failure disease caused by aortic constriction.

3. Use according to claim 1, characterized in that: The resistin RELMα achieves treatment of heart failure disease caused by aortic constriction by simultaneously inhibiting cardiac fibrosis and promoting cardiovascular neogenesis.

4. Use according to claim 1, characterized in that: The drug includes: resistin RELMα as an active ingredient, and a pharmaceutically acceptable solvent, an excipient, and / or a carrier.

5. Use according to claim 1 or 4, characterized in that: The injection preparation is an injection preparation by intramyocardial injection.

6. Use according to claim 1 or 4, characterized in that: The injection preparation is an injection preparation by subcutaneous injection.

7. Use according to claim 1 or 4, characterized in that: The injection preparation is an injection preparation by intramuscular injection.

8. Use according to claim 1 or 4, characterized in that: The injection preparation is an injection preparation by intravenous injection.

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