Application of resistin in preparation of medicine for treating heart diseases

Abstract

The invention relates to the field of biological medicine, and discloses application of resistin in preparation of a medicine for treating heart diseases. The resistin is found to have the effects of inhibiting cardiac fibrosis and promoting angiogenesis for the first time, so that the resistin has the potential of preparing the medicine for treating heart diseases (cardiovascular diseases), and meanwhile, a new theoretical basis and a potential intervention target are expected to be provided for heart failure.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to the application of resistin in the preparation of heart disease medicines. Background Art

[0002] Cardiovascular disease is the leading cause of death from disease worldwide. Unhealthy lifestyles have led to a huge number of people with cardiovascular risk factors. At the same time, the aging of the population is accelerating, and cardiovascular disease remains one of the biggest threats to residents' health. Tissue remodeling after cardiac injury is believed to be the main pathological mechanism leading to decreased cardiac function. Manifestations of cardiac tissue remodeling include activation of the renin-angiotensin-aldosterone system, fibrosis, and decreased vascular density. Existing clinical treatment measures include drugs that mainly reduce the cardiac load by inhibiting the renin-angiotensin-aldosterone system, thereby delaying cardiac remodeling. How to regulate the process of fibrosis after cardiac injury and promote cardiac angiogenesis is an urgent problem to be solved in the field of cardiovascular disease research.

[0003] Heart diseases including myocardial infarction, pathological hypertrophy, and heart failure often manifest as aseptic injury. Aseptic injury can lead to a series of inflammatory responses. Tissue-resident macrophages play the role of "immune surveillance" with the ultimate goal of restoring damaged tissue to homeostasis. Cardiac tissue is rich in immune cells, and the number of immune cells it contains is more than 12 times that of skeletal muscle. Most of these immune cells are macrophages, which were previously considered to be a relatively uniform cell population that highly expresses RELMα.

[0004] RELMα belongs to the resistin family. Human resistin is very similar to mouse RELMα in origin and function, that is, both are mainly secreted by mononuclear macrophages and have the function of regulating metabolism and immunity. Previous studies have suggested that RELMα plays an important role in metabolic homeostasis and inflammation. At present, there are no literature reports on the role of resistin in protecting tissue remodeling after cardiac injury, especially the role of RELMα in inhibiting cardiac fibrosis and promoting cardiovascular regeneration. Summary of the invention

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

[0006] The specific technical scheme of the present invention is: In a first aspect, the present invention provides the use of resistin in the preparation of heart disease drugs.

[0007] As described in the background technology section of this application, in the prior art, the role of resistin in regulating metabolism and inflammation in many species including humans is relatively clear. Currently, there are no reports internationally on the role of resistin in the heart, especially in heart failure. The present invention proposes for the first time that resistin can inhibit fibrosis and promote angiogenesis, and is expected to provide a new theoretical basis and potential intervention target for heart failure. Specifically, the present invention treats a heart failure model with resistin and finds that resistin has the ability to reduce fibrosis and promote angiogenesis, thereby having the potential to prepare heart disease (cardiovascular disease) drugs.

[0008] Preferably, the heart disease is heart failure.

[0009] Preferably, the heart disease drug comprises: resistin as an active ingredient, and pharmaceutically acceptable solvents, excipients and / or carriers.

[0010] More preferably, the heart disease drug is an injectable preparation or an oral preparation.

[0011] More preferably, the injection preparation is an injection preparation for intramyocardial injection, subcutaneous injection, intramuscular injection or intravenous injection.

[0012] In a second aspect, the present invention provides the use of resistin in the preparation of anti-fibrosis drugs.

[0013] Preferably, the anti-fibrosis drug is a drug for treating fibrotic diseases or a tool drug for scientific research.

[0014] Preferably, the fibrosis is cardiac fibrosis.

[0015] Preferably, the anti-fibrosis drug comprises: resistin as an active ingredient, and pharmaceutically acceptable solvents, excipients and / or carriers.

[0016] Preferably, the anti-fibrosis drug is an injectable preparation or an oral preparation.

[0017] More preferably, the injection preparation is an injection preparation for intramyocardial injection, subcutaneous injection, intramuscular injection or intravenous injection.

[0018] In a third aspect, the present invention provides a method for inhibiting cardiac fibroblast fibrosis in vitro: cardiac fibroblasts are cultured in vitro, and resistin is added to inhibit the expression levels of collagen protein, periostin protein and a-SMA protein, thereby inhibiting cardiac fibroblast fibrosis.

[0019] The present invention found through in vitro experiments that resistin can significantly reduce the expression level of fibrosis markers of cardiac fibroblasts, thus indicating that it has the effect of inhibiting fibrosis of cardiac fibroblasts.

[0020] In a fourth aspect, the present invention provides the use of resistin in the preparation of angiogenesis promoters.

[0021] Preferably, the angiogenesis promoter is a drug used to treat a disease or a tool drug used in scientific research.

[0022] Preferably, the blood vessel is a cardiac vessel.

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

[0024] Preferably, the angiogenesis promoter is an injectable preparation or an oral preparation.

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

[0026] In a fifth aspect, the present invention provides a method for promoting the tube formation and transformation of venous endothelial cells into new blood vessels under in vitro conditions: venous endothelial cells are cultured under in vitro conditions, and resistin is added to promote the tube formation and transformation of venous endothelial cells into new blood vessels.

[0027] The present invention found through in vitro experiments that resistin can significantly increase the number of tubes formed by venous endothelial cells, thereby indicating that it has the effect of promoting angiogenesis.

[0028] Compared with the prior art, the beneficial effects of the present invention are: the present invention is the first to discover that resistin has the effect of inhibiting cardiac fibrosis and promoting cardiovascular regeneration, thereby having the potential to prepare heart disease (cardiovascular disease) drugs, and is expected to provide a new theoretical basis and potential intervention targets for heart failure. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is a comparison of the cardiac function indicators of mice evaluated by ultrasound Doppler in the sham group (sham group), control group (TAC), and resistin (RELMα) administration group (TAC+RELMα) after aortic constriction model surgery 9 weeks after surgery; where: A is the left ventricular septal thickness in diastole (LVID; d); 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.

[0030] Figure 2After the aortic constriction model was performed on mice, heart tissues were collected from the sham group (sham group), control group (TAC), and resistin (RELMα) administration group (TAC+RELMα) 9 weeks after surgery for fibrosis and angiogenesis detection. A is a Masson staining image of fibrosis; scale: 300 um; B is the quantitative result of evaluating cardiac fibrosis; C. is a CD31 staining image; scale: 100 um; D is the quantitative result of cardiac blood vessels; * P<0.05; **P<0.01; ***P<0.001.

[0031] Figure 3 The figure shows the comparison of various indicators of rat fibroblasts cultured in vitro and added with different concentrations of RELMα (0, 1, 2, 4, 8 ug / ml); A is the Western blot detection of fibrosis indicators Collagen I, Periostin and a-SMA bands; 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.

[0032] 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 for culture. Among them: A is the cell images captured 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 4 hours; D is the quantitative result of the number of endothelial cell tubes after 6 hours; E is the quantitative result of the number of endothelial cell tubes after 12 hours. Scale bar: 200 um; * P<0.05; **P<0.01; ***P<0.001. DETAILED DESCRIPTION

[0033] The present invention will be further described below in conjunction with the embodiments.

[0034] Overall embodiment In a first aspect, the present invention provides the use of resistin in the preparation of heart disease drugs.

[0035] In some preferred embodiments, the heart disease is heart failure.

[0036] In some preferred embodiments, the heart disease drug includes: resistin as an active ingredient, and pharmaceutically acceptable solvents, excipients and / or carriers.

[0037] In some preferred implementation cases, the heart disease drug is an injectable preparation and an oral preparation.

[0038] In some more preferred embodiments, the injection preparation is an injection preparation that is injected intramyocardially, subcutaneously, intramuscularly or intravenously.

[0039] In a second aspect, the present invention provides the use of resistin in the preparation of anti-fibrosis drugs.

[0040] In some preferred implementation cases, the anti-fibrosis drug is a drug for treating fibrotic diseases or a tool drug for scientific research.

[0041] In some preferred embodiments, the fibrosis is cardiac fibrosis.

[0042] In some preferred implementation cases, the anti-fibrosis drug includes: resistin as an active ingredient, and pharmaceutically acceptable solvents, excipients and / or carriers.

[0043] In some preferred implementation cases, the anti-fibrosis drug is an injectable preparation or an oral preparation.

[0044] In some more preferred embodiments, the injection preparation is an injection preparation that is injected into the myocardium, intradermally, subcutaneously, intramuscularly or intravenously.

[0045] In a third aspect, the present invention provides a method for inhibiting cardiac fibroblast fibrosis in vitro: cardiac fibroblasts are cultured in vitro, and resistin is added to inhibit the expression levels of collagen protein, periostin protein and a-SMA protein, thereby inhibiting cardiac fibroblast fibrosis.

[0046] In a fourth aspect, the present invention provides the use of resistin in the preparation of angiogenesis promoters.

[0047] In some preferred implementation cases, the angiogenesis promoter is a drug used to treat a disease or a tool drug used in scientific research.

[0048] In some preferred embodiments, the blood vessel is a cardiac vessel.

[0049] In some preferred embodiments, the angiogenesis promoter includes: resistin as an active ingredient, and a pharmaceutically acceptable solvent, excipient, and / or carrier.

[0050] In some preferred implementation cases, the angiogenesis promoter is an injectable preparation or an oral preparation.

[0051] In some more preferred embodiments, the injection preparation is an injection preparation that is injected intramyocardially, subcutaneously, intramuscularly or intravenously.

[0052] In a fifth aspect, the present invention provides a method for promoting the tube formation and transformation of venous endothelial cells into new blood vessels under in vitro conditions: venous endothelial cells are cultured under in vitro conditions, and resistin is added to promote the tube formation and transformation of venous endothelial cells into new blood vessels. Specific embodiments Example 1 (1) Construction of mouse heart failure model: After 8-10 week old mice (Shanghai Slake Biotechnology Co., Ltd.) were anesthetized with isoflurane gas, the skin and muscles were separated and the suprasternal fossa was approached. Part of the sternum was cut along the midline of the sternum. The visual field was exposed using a small animal chest brace. The aortic arch was found and bypassed with a 6-0 suture. A 27 gauge padding needle was placed horizontally on the aortic arch and tied, and then the padding needle was removed. The sternum and skin were sutured and placed on a warming pad until the mouse woke up. Mice that underwent the same surgical procedure but without knotting were used as the sham group. Drug administration group: Resistin RELMα (Peprotech Cat#450-26; 10 mg / mouse) was intraperitoneally administered starting from the 6th week after surgery, twice a week for 4 consecutive weeks. The control group was injected with the corresponding solvent (PBS solution containing 0.1% BSA).

[0054] (2) Doppler ultrasound was used to evaluate the cardiac function of mice before and after surgery.

[0055] (3) Sample collection: 4 weeks after resistin injection, CO 2 Mice were anesthetized and killed, and the hearts were collected. The heart tissues were fixed, embedded in OCT, and frozen, and the degree of neovascularization and fibrosis was quantitatively evaluated using CD31 antibody (BD Cat# 550274) and Masson staining kit (Solarbio), respectively, and quantitative analysis was performed using imageJ software.

[0056] (4) Figure 1 This is a comparison chart of various cardiac function indicators in different groups (sham group, control group (TAC), resistin (RELMα) administration group (TAC+RELMα)) after aortic constriction model was performed on mice; among them: A is the left ventricular septal thickness during diastole (LVID; d); B is the left ventricular ejection fraction (LVEF); C is the left ventricular short axis shortening rate (FS); Figure 2 Fibrosis and angiogenesis were detected after sampling heart tissue 9 weeks after surgery. A is a Masson staining image of fibrosis; B is a quantitative result of evaluating cardiac fibrosis; C is a CD31 staining image; D is a quantitative result of cardiac blood vessels.

[0057] like Figure 1As shown in the figure: 9 weeks after surgery, the cardiac systolic function indicators LVEF (51.5%) and FS value (26.7%) of the control group had decreased significantly, while the LVEF and FS values ​​of the RELMα administration group were maintained at around 64.0% and 34.5%, indicating that the cardiac systolic function was significantly improved compared with the control group. Figure 2 As shown, the vascular density staining quantitative of the drug-treated group (6.6%) was significantly increased compared with the control group (4.8%), and the fibrosis area of ​​the RELMα group (2.1%) was significantly reduced compared with the control group (5.9%). The above results show that in the mouse heart failure model, supplementation of resistin RELMα can improve cardiac function by inhibiting fibrosis and promoting angiogenesis.

[0058] Example 2 (1) P0 newborn rat fibroblasts were isolated using a rat heart dissociation kit (Miltenyi, No. 130-098-373). These fibroblasts were then transferred to a 10 cm culture dish and cultured overnight in a low-glucose DMEM medium containing 10% serum.

[0059] (2) On the second day, the fibroblasts were digested with trypsin solution and seeded on 6-well plates, and cultured again overnight in low-glucose DMEM medium containing 10% serum. On the third day, they were switched to serum-free low-glucose medium for further culture.

[0060] (3) After 24 hours of incubation, the culture medium was changed to culture medium supplemented with 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) and culture continued.

[0061] (4) After 24 hours of drug treatment, protein was extracted using RIPA lysis buffer (Beyotime). Protein samples (50ug) were separated using 10% or 15% SDS-PAGE gel electrophoresis and transferred to PVDF membranes (Miltenyi). After blocking with 5% BSA solution, primary antibody was added and incubated overnight at 4°C; the next day, after washing with PBST, HRP secondary antibody was incubated at room temperature for 1 hour, and after washing with PBST, ECL reagent (Merck Millipore) was used for color development and exposed using Amersham Image Quant800 imager. Quantitative analysis was performed using imageJ software. Primary antibody information: Anti-mPeriostin / OSF-2 Goat IgG (R&D Company Cat# AF2955); Recombinant Anti-Collagen I antibody (Abcom Company Cat# ab270993); Rabbit polyclonal to alpha smooth muscle Actin (Abcom Company Cat# ab5694); HRP Conjugated Anti-GAPDH Recombinant Antibody (Huabio Company Cat# ET1702-66). Secondary antibody information: HRP Conjugated Goat anti-Rabbit IgG Goat Polyclonal Antibody (Huabio Company Cat# HA1001) Donkey Anti-Goat IgG H&L (HRP) (Abcam Company Cat# ab6885).

[0062] (4) The results are as follows Figure 3 As shown: the collagen protein in the 2-8 ug / ml RELMα group was significantly lower than that in the simple TGFβ model group; the Periostin protein in the 2ug / ml RELMα group was significantly lower than that in the simple TGFβ model group; the a-SMA protein in the 4ug / ml RELMα group was significantly lower than that in the simple TGFβ model group. The above results show that supplementing RELMα can inhibit the fibrosis process of fibroblasts.

[0063] Example 3 (1) Add 50ul / well of Matrigel (Corning) melted overnight into a 96-well plate. Place the 96-well plate with Matrigel in a cell culture incubator and incubate at 37°C for 30-60 minutes.

[0064] (2) Well-growing human umbilical vein endothelial cells (HUVECs) were digested with Accutase and resuspended in endothelial cell complete medium containing 1% serum.

[0065] (3) Resuspend the cells in culture medium containing different concentrations of RELMα (0 ug / ml, 1 ug / ml, 2 ug / ml, 4 ug / ml, 8 ug / ml); culture the cells at 1.7×10 4 The cells were transferred to a 96-well plate at a density of / 100ul for culture.

[0066] (4) The 96-well plate was placed in a cell culture incubator. Cell images were captured using a bright-field inverted microscope at 2.5, 4, 6, and 12 hours. ImageJ software was used to quantitatively analyze the number of angiogenesis.

[0067] (5) The results are as follows Figure 4 As shown: at 2.5 hours (h), the number of angiogenesis in the 4-8 ug / ml RELMα group was significantly increased compared with the control group; at 4 h, the number of angiogenesis in the 4ug / ml RELMα group was significantly increased compared with the control group; at 6 h, the number of angiogenesis in the 4ug / ml RELMα group was significantly increased compared with the control group; at 8 h, the number of angiogenesis in the 4ug / ml RELMα group was significantly increased compared with the control group. The above results show that supplementing RELMα can inhibit the fibrosis process of fibroblasts.

[0068] The experimental results of Examples 1 to 3 above show that the beneficial effects of the present invention are as follows: the present invention is the first to discover that resistin has the effects of inhibiting cardiac fibrosis and promoting cardiovascular regeneration, thereby having the potential to prepare heart disease (cardiovascular disease) drugs, and is expected to provide a new theoretical basis and potential intervention targets for heart failure.

[0069] The raw materials and equipment used in the present invention, unless otherwise specified, are all commonly used raw materials and equipment in the art; the methods used in the present invention, unless otherwise specified, are all conventional methods in the art.

[0070] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any simple modification, change and equivalent transformation made to the above embodiment based on the technical essence of the present invention still falls within the protection scope of the technical solution of the present invention.

Claims

1. Application of resistin in the preparation of drugs for heart diseases.

2. The use according to claim 1, characterized in that: The heart disease medicine comprises: resistin as an active ingredient, and pharmaceutically acceptable solvents, excipients and / or carriers.

3. Application of resistin in the preparation of anti-fibrosis drugs.

4. The use according to claim 3, characterized in that: The anti-fibrosis drug is a drug used to treat fibrotic diseases or a tool drug used in scientific research.

5. The use according to claim 3 or 4, characterized in that: The fibrosis is cardiac fibrosis.

6. A method for inhibiting cardiac fibroblast fibrosis in vitro, characterized in that: Cardiac fibroblasts were cultured in vitro and resistin was added to inhibit the expression levels of collagen protein, periostin protein and a-SMA protein, thereby inhibiting the fibrosis of cardiac fibroblasts.

7. Application of resistin in the preparation of angiogenesis promoters.

8. The use according to claim 7, characterized in that: The angiogenesis promoter is a drug used to treat diseases or a tool drug used in scientific research.

9. The use according to claim 7 or 8, characterized in that: The blood vessels are cardiovascular vessels.

10. A method for promoting the transformation of venous endothelial cells into new blood vessels in vitro, characterized in that: Venous endothelial cells were cultured in vitro and resistin was added to promote the formation of tubes in the venous endothelial cells, thereby converting them into new blood vessels.

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