CN113350346B

CN113350346B - Use of vincristine in preventing or treating myocardial fibrosis

Info

Publication number: CN113350346B
Application number: CN202110609879.XA
Authority: CN
Inventors: 何燕; 葛晨亮
Original assignee: First Affiliated Hospital of Guangxi Medical University
Current assignee: First Affiliated Hospital of Guangxi Medical University
Priority date: 2021-06-01
Filing date: 2021-06-01
Publication date: 2023-06-27
Anticipated expiration: 2041-06-01
Also published as: CN113350346A

Abstract

The invention discloses application of vincristine in preventing or treating myocardial fibrosis, and the vincristine or a medicament formed by pharmaceutically acceptable salts, esters or solvates thereof is proved to have obvious inhibition effect on heart reconstruction by experiments in vivo and experiments performed on cells in vitro in rats; vincristine has obvious therapeutic effect on myocardial fibrosis and can inhibit myocardial fibrosis.

Description

Use of vincristine in preventing or treating myocardial fibrosis

Technical Field

The invention belongs to the technical field of vincristine application, and particularly relates to application of vincristine in preventing or treating myocardial fibrosis.

Background

Cardiovascular disease threatens human health and has become a major public health concern worldwide. Myocardial fibrosis is a common pathological change that occurs from various cardiovascular diseases to a certain stage, and various cardiovascular diseases including hypertension, coronary heart disease and atrial fibrillation can lead to myocardial fibrosis, cause dysfunction of systole and diastole, and finally lead to heart failure. Currently, few drugs are clinically available for the treatment of myocardial fibrosis. Therefore, how to prevent and reverse myocardial fibrosis is an important point and difficulty in urgent research in the cardiovascular field.

After damage to heart tissue, the immune system in the body is activated and a wound healing response is triggered, which is a defensive mechanism of the body, and recruitment and infiltration of inflammatory immune cells such as monocytes, macrophages and neutrophils in necrotic areas are promoted. These activated inflammatory cells secrete a large number of inflammatory and pro-fibrotic factors, stimulating the differentiation of fibroblasts into myofibroblasts, which synthesize and secrete excessive extracellular matrix leading to fibrosis, the major structural proteins of which are fibrous collagens, including type I collagen (collagani) and type III collagen (collagani).

Immune inflammatory response plays an important role in the formation and progression of myocardial fibrosis, innate immunity is the first line of defense of the body against exogenous pathogens and various lesions, and inflammatory corpuscles are an important part of the body's innate immunity. NLRP3 (nucleic acid-binding oligomerization domain leucine-rich repeat and pyrin domains-containing protein 3) is one of the most in-depth inflammatory bodies studied, NLRP3 inflammatory bodies are polyprotein complexes composed of NLRP3, apoptosis-related speckle-like protein (ASC) containing a C-terminal caspase recruitment domain and caspase-1 precursor (pro-caspase-1). After myocardial injury caused by various cardiovascular diseases, necrotic cardiomyocytes produce active oxygen, causing potassium outflow and ATP release, which activate NF (nuclear factor) - κb and TLR (toll-like receptor) -mediated signaling pathways, inducing up-regulation of NLRP3 inflammatory corpuscle component expression, initiating a sterile inflammatory response. NLRP3 remains in a state of long-term activation and releases interleukin 1 (IL-1β) and interleukin 18 (IL-18) for a long period of time in the presence of cardiovascular injury factors, gradually leading to the formation of cardiac structural remodeling and fibrosis. More and more studies confirm that NLRP3 inflammasome is a key mediator of the progression of fibrosis due to cardiovascular disease, and is a new molecular target for treating myocardial fibrosis.

Vincristine is an important secondary metabolite extracted from the herb vinca rosea. Molecular formula C ₄₆ H ₅₆ N ₄ O ₁₀ At present, the traditional Chinese medicine composition is mainly used for treating acute lymphoblastic leukemia, hodgkin's disease, breast cancer, malignant lymphoma, small cell lung cancer and other diseases clinically. The prevention and treatment effect of vincristine on myocardial fibrosis is not reported.

Disclosure of Invention

In view of the above-mentioned shortcomings of the prior art, the present invention provides a medicament effective in preventing or treating myocardial fibrosis, and provides a novel use of vincristine. Vincristine can be used for preventing or treating myocardial fibrosis.

The invention provides a medicine composed of vincristine or pharmaceutically acceptable salt, ester or solvate thereof, and application of the medicine in preparing medicines for preventing or treating myocardial fibrosis.

A medicine comprising vincristine or pharmaceutically acceptable salt, ester or solvate thereof can effectively prevent or treat myocardial fibrosis after dissolving vincristine in vivo.

The myocardial fibrosis generally refers to a heart pathological change of myocardial tissue, which is caused by chronic inflammation and apoptosis of myocardial tissue and is characterized by excessive activation of fibroblasts in myocardial tissue, secretion of a large amount of collagen fibers and excessive deposition, and obvious increase of collagen concentration and collagen volume fraction, and imbalance and arrangement of various types of collagen proportion.

The salt is any one of vincristine sulfate, vincristine folic acid and vincristine succinate.

Further, the medicine is a medicine preparation consisting of effective dose of vincristine and pharmaceutically acceptable auxiliary materials; the auxiliary materials are selected from one or more of the following groups of substances: microcrystalline cellulose, pregelatinized starch, lactose, magnesium stearate, talc, beta cyclodextrin, sodium bicarbonate and croscarmellose sodium.

Further, the vincristine is used in an amount of: 25ug/kg;

further, the dosage form of the pharmaceutical preparation is a liquid or solid dosage form;

further, the administration route of the pharmaceutical preparation is injection administration.

The invention has the beneficial effects that:

the invention develops the therapeutic effect of vincristine on myocardial fibers through in-vivo experiments and in-vitro cell experiments of rats, and results prove that: vincristine has obvious inhibiting effect on heart reconstruction; vincristine has obvious therapeutic effect on myocardial fibrosis and can inhibit myocardial fibrosis.

Drawings

FIG. 1 is a chemical structural formula of vincristine in accordance with the present invention;

FIG. 2 is a graphical representation of the results of a Heart index (Heart index) determination of the present invention;

FIG. 3 is a graphical representation of the results of the degree of myocardial fibrosis of the present invention;

FIG. 4 is a schematic representation of the results of immunohistochemical detection of myocardial CollagenI, collagenIII of the present invention;

FIG. 5 is a schematic diagram showing the results of immunohistochemical detection of myocardial interleukin 1 (IL-1. Beta.) and interleukin 18 (IL-18) expression in accordance with the present invention;

FIG. 6 is a schematic diagram showing the result of Western blot (Western blot) detection of IL-1 beta and IL-18 protein expression in myocardial tissue;

FIG. 7 is a schematic diagram showing the results of Western blot (Western blot) detection of expression levels of ASC, NLRP3, pro-Caspase-1, caspase-1-p20 proteins in myocardial tissue;

FIG. 8 is a graph showing the detection of vincristine toxicity to cardiac fibroblasts by CCK8 of the present invention. Results of enzyme-linked immunosorbent assay (Elisa) for detecting IL-1 beta, IL-18 concentration in cardiac fibroblast supernatant;

FIG. 9 is a schematic diagram showing the results of Western blot (Western blot) detection of expression levels of ASC, NLRP3, pro-Caspase-1, caspase-1-p20 proteins in cells.

Detailed Description

The invention is further described below with reference to fig. 1-9:

rat in vivo experiment and in vitro cell experiment

Raw materials: 32 SPF-grade male SD rats, with a body weight of 200+ -20 g, purchased from university of medical university of Guangxi; vincristine sulfate as a white powder was dissolved in physiological saline, purchased from Soy Corp (2068-78-2), and isoprenaline hydrochloride as a white powder was dissolved in physiological saline, purchased from Sigma-Aldrich (I5627) in the United states. The antibodies used: collagen I (Proteintech, 14695-1-AP), collagen III (Proteintech, 22734-1-AP), NLRP3 (NOVUS, NBP 2-12446), ASC (Cell Signaling Technology, 67824), pro-caspase1 (Abcom, ab 179515), clear-caspase 1-p20 (Proteintech, 22915-1-AP), IL-18 (ab 71495; abcam), IL-1β (31202;Cell Signaling Technology), GAPDH (Proteintech, 60004-1-Ig, 1:5000). Rat interleukin 1 beta (IL-1 beta) ELISA kit (China bioengineering company), and rat interleukin 18 (IL-18) ELISA kit (China bioengineering company).

Model construction and animal experiments: rats were randomized into control (n=8), myocardial fibrosis model (n=8), vincristine low dose (25 ug/kg, n=8) and vincristine high dose (50 ug/kg, n=8). Isoprenaline injection replicates the rat myocardial fibrosis model. The vincristine high and low dose groups were previously given subcutaneous injections of VCR25ug/kg,50ug/kg, respectively, in the morning of the day, and the control group and the myocardial fibrosis group were injected with an equal amount of physiological saline. The myocardial fibrosis group and the vincristine high and low dose group are subcutaneously injected with 5mg/kg isoprenaline, and the control group is injected with an equivalent amount of physiological saline. During which drinking water was taken normally for 10 consecutive days. Rats were sacrificed on day eleven and each index was tested. Remarks: the addition amount of vincristine, the rat was converted according to the vincristine content of vincristine sulfate, i.e. 824.96/923.0361, by administering 25 ug/50 ug/kg of vincristine (low dose group) per kg of vincristine (high dose group).

1. Cardiac weight and cardiac index

The heart weights, body weights and calculated heart index (heart weight/body weight) of the rats of each group were measured after the end of the intervention. The appearance of panel a of fig. 2 shows that heart is more hypertrophic in the myocardial fibrosis group than in the control group, suggesting successful molding of the myocardial fibers, while the vincristine high-dose and low-dose groups show a reduced heart volume than the myocardial fibrosis group. The measured cardiac index (cardiac mass/rat body weight) of each group of rats is shown in panel B of fig. 2, the cardiac index value of the model group is significantly higher than that of the control group, and the cardiac index value of the vincristine high and low dose group is lower than that of the model group, so that the inhibition of cardiac remodeling by vincristine is confirmed (P < 0.05).

2. Hematoxylin-eosin (HE) staining and Masson staining to observe histological changes and the extent of myocardial fibrosis

Rat ventricular tissue was fixed in 10% formaldehyde solution, paraffin embedded and sectioned, and hematoxylin-eosin (HE) staining and Masson staining were performed to observe tissue changes and the degree of myocardial fibrosis. Fibrous tissue appeared blue under Masson staining. The HE staining results in the A graph of FIG. 3 show that the control cardiomyocytes were normally arranged, the myocardial fibers were neat, no breaks, and the shapes were substantially consistent; myocardial fibrosis groups can be seen with cardiomyocyte lysis, myocardial fibrosis disorder, inflammatory cell infiltration; the damage of myocardial fiber of a vincristine administration group is reduced, and infiltration of inflammatory cells is reduced. The Masson staining results showed: the normal control group can see that red myocardial cells are arranged neatly and compactly, and a small amount of blue collagen fibers are distributed in the interstitial of the myocardium in a scattered manner; the myocardial fibrosis group can be seen to be deposited with a large amount of blue collagen around the myocardial stroma and blood vessels, and has the characteristics of disturbance trend and crisscross; the high and low dose groups of vincristine have obvious cardiovascular and interstitial collagen deposition, and compared with the model group, the degree of fibrosis is obviously reduced. The ratio of myocardial fibrosis area to total area was calculated for each group of rats by Image J software in panel B of fig. 3. Compared with the control group, the myocardial fibrosis group is obviously increased, the successful molding of myocardial fibrosis is confirmed, the ratio of the high-dose and low-dose group fibrosis areas of vincristine is obviously reduced, and the treatment effect (P < 0.05) of vincristine on myocardial fibrosis is confirmed.

3. Immunohistochemical detection of expression of CollagenI, collagenIII in myocardial tissue

The graph a of fig. 4 shows that the expression of CollagenI, collagenIII in the cardiac muscle matrix in the cardiac muscle fibrosis group was significantly increased compared to the control group, confirming that the cardiac muscle fibrosis modeling was successful. The expression of vincristine was reduced in the high and low dose group CollagenI, collagenIII compared to the myocardial fibrosis group. Panel B of fig. 4 shows similar data statistics from Image J software, further demonstrating that vincristine can inhibit myocardial fibrosis (< P < 0.05).

4. Immunohistochemistry detects the expression of interleukin 1 (IL-1. Beta.) and interleukin 18 (IL-18) in myocardial tissue. The expression of IL-1. Beta. And IL-18 in the myocardial fibrosis group is significantly increased compared to the control group shown in panel A of FIG. 5. The expression of IL-18 was reduced in the high and low dose groups of vincristine IL-1β compared to the myocardial fibrosis group. The same conclusion was drawn by Image J software, further confirming that vincristine reduced IL-1 β, IL-18 expression in myocardial tissue (< 0.05 with P).

5. Western blot (Western blot) is used for detecting the expression level of IL-1 beta and IL-18 protein in myocardial tissue. The expression level of IL-1 β, IL-18 protein in the myocardial fibrosis model group is significantly increased compared to the normal group (P < 0.05) shown in panel a of fig. 6. Compared with the model group, the expression level of IL-1 beta and IL-18 protein in the high and low dose groups of vincristine is obviously reduced (P is less than 0.05). The same conclusion was reached by image j software analysis in panel B, C of fig. 6, further confirming that vincristine can reduce IL-1 beta, IL-18 expression in myocardial tissue (< 0.05 by P).

6. Western blot (Western blot) was performed to determine the expression levels of ASC, NLRP3, pro-Caspase-1, caspase-1-p20 proteins in myocardial tissue. The significantly increased expression level of NLRP3, caspase-1-P20 protein (< 0.05) in the myocardial fibrosis model group compared to the normal group is shown in FIG. 7, confirming that NLRP3 inflammatory corpuscles are activated in the heart. Compared with the model group, the expression level of Caspase-1-P20 protein in the high and low dose groups of vincristine is significantly reduced (P < 0.05), and the combination of the vincristine reduces the expression of IL-1 beta and IL-18 in myocardial tissues, thus confirming that the vincristine can improve myocardial fibrosis by inhibiting activation of NLRP3 inflammatory corpuscles (P < 0.05).

7. The inhibition of NLRP3 activation by vincristine was further confirmed by in vitro experiments. Primary cultured rat cardiac fibroblasts, cells were identified by immunofluorescence detection of the expression of the fibroblast marker protein, vimentin, which was expressed positively in FIG. 8, panel A, confirming that the cultured cells were fibroblasts. The toxicity of vincristine to cardiac fibroblasts was studied by Cell Counting Kit-8 (CCK 8), and it was shown in FIG. 8B that vincristine at a concentration ranging from 10 to 100umol/L was not significantly toxic to cardiac fibroblasts, so that 10, 20, 50umol/L vincristine was selected for the next experiment. Stimulation of fibroblasts with Lipopolysaccharide (LPS) +adenosine triphosphate (ATP) to activate NLRP3 inflammatory bodies, addition of vincristine to investigate whether vincristine can inhibit activation of heart fibroblast NLRP3 inflammatory bodies, and detection of IL-1 beta, IL-18 concentration in cell supernatants was performed by enzyme-linked immunosorbent assay (Elisa). The concentration of IL-1 beta and IL-18 in the cell supernatant was significantly increased in the LPS+ATP-stimulated group compared to the control group, confirming that NLRP3 inflammatory corpuscles in fibroblasts were activated after LPS+ATP stimulation. In contrast to the LPS+ATP stimulated group, the concentration of IL-1. Beta. And IL-18 in the cell supernatants of the

vincristine

10, 20 and 50umol/L groups was significantly reduced, demonstrating that vincristine inhibits activation of NLRP3 inflammatory corpuscles in cardiac fibroblasts (< 0.05. Times.P).

8. Western blot (Western blot) was used to detect the expression levels of ASC, NLRP3, pro-Caspase-1, caspase-1-p20 proteins in cells. The significantly increased expression level of NLRP3, caspase-1-P20 protein (< 0.05) in LPS+ATP stimulated group compared to normal group is shown in FIG. 9, confirming that NLRP3 inflammatory corpuscles are activated in fibroblasts. The expression level of Caspase-1-P20 protein was significantly reduced (P < 0.05) in the vincristine 10umol/L,20umol/L,50umol/L groups compared to the LPS+ATP stimulated group, and the combination of vincristine above reduced the concentration of IL-1 beta, IL-18 in the cell supernatant, confirming that vincristine can inhibit activation of NLRP3 inflammatory corpuscles in cardiac fibroblasts (P < 0.05).

Those skilled in the art will recognize that numerous variations to the above description are possible, and that the examples are intended only to be illustrative of one or more particular implementations.

While there have been described and illustrated what are considered to be example embodiments of the present invention, it will be understood by those skilled in the art that various changes and substitutions can be made therein without departing from the spirit of the invention. In addition, many modifications may be made to adapt a particular situation to the teachings of the invention without departing from the central concept thereof as described herein. Therefore, it is intended that the invention not be limited to the particular embodiment disclosed, but that the invention will include all embodiments falling within the scope of the invention and equivalents thereof.

Claims

1. The use of vincristine or a pharmaceutically acceptable salt thereof as the sole active ingredient in the manufacture of a medicament for the treatment of myocardial fibrosis, characterized in that the pharmaceutically acceptable salt of vincristine is vincristine sulfate.

2. The use according to claim 1, wherein the medicament is a pharmaceutical preparation consisting of an effective amount of vincristine or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable adjuvant.

3. The use according to claim 2, wherein the auxiliary material is selected from one or more of the following group of substances: microcrystalline cellulose, pregelatinized starch, lactose, magnesium stearate, talc, beta cyclodextrin, sodium bicarbonate and croscarmellose sodium.

4. The use according to claim 2, wherein the pharmaceutical formulation is in the form of a liquid or solid dosage form.

5. The use according to claim 2, wherein the route of administration of the pharmaceutical formulation is injection.