CN113350346B - Use of vincristine in preventing or treating myocardial fibrosis - Google Patents

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

Application of Vincristine in Prevention or Treatment of Myocardial Fibrosis

technical field

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

Background technique

Cardiovascular disease threatens human health and has become a major public health issue of global concern. Myocardial fibrosis is a common pathological change in the development of various cardiovascular diseases to a certain stage. Various cardiovascular diseases, including hypertension, coronary heart disease, and atrial fibrillation, can lead to myocardial fibrosis, causing cardiac systolic and diastolic dysfunction, and eventually Will cause heart failure. At present, there are few drugs available clinically for the treatment of myocardial fibers. Therefore, how to prevent and reverse myocardial fibrosis is an urgent research focus and difficulty in the cardiovascular field.

After injury to heart tissue, the immune system in the body is activated and a wound healing response is triggered, which is a defense mechanism of the body and promotes the proliferation of inflammatory immune cells such as monocytes, macrophages and neutrophils in the necrotic area recruitment and infiltration. These activated inflammatory cells secrete a large number of inflammatory and pro-fibrotic factors, stimulating fibroblasts to differentiate into myofibroblasts, myofibroblasts will synthesize and secrete excessive extracellular matrix and lead to fibrosis, the extracellular matrix The main structural protein is fibrous collagen, including type I collagen (CollagenI) and type III collagen (CollagenIII).

The immune inflammatory response plays an important role in the formation and progression of myocardial fibrosis. Innate immunity is the body's first line of defense against exogenous pathogens and various injuries, and inflammasomes are an important part of the body's innate immunity. NLRP3 (nucleotide-binding oligomerization domain leucine-rich repeat and pyrindomains-containingprotein 3) is the most deeply studied inflammasome. The apoptosis-related speck-like protein (apoptosis-related speck-like protein containing a C-terminal caspase recruitment domain, ASC) and caspase-1 precursor (pro-caspase-1). After myocardial injury caused by various cardiovascular diseases, necrotic cardiomyocytes will produce reactive oxygen species, causing the outflow of potassium ions and the release of ATP, and these signals activate NF (nuclearfactor)-κB and TLR (toll-like receptor)-mediated signaling pathways , Inducing the up-regulation of the expression of NLRP3 inflammasome components, initiating a sterile inflammatory response. Under the condition of persistent cardiovascular injury factors, NLRP3 maintains a long-term activation state and releases interleukin 1 (IL-1β) and interleukin 18 (IL-18) for a long time, which gradually leads to cardiac structural remodeling and fibrosis formation. More and more studies have confirmed that NLRP3 inflammasome is a key mediator in the progression of cardiovascular disease leading to fibrosis, and it is a new molecular target for the treatment of myocardial fibrosis.

Vincristine is an important secondary metabolite extracted from the herbaceous plant periwinkle. The molecular formula is C ₄₆ H ₅₆ N ₄ O ₁₀ , and it is mainly used clinically to treat diseases such as acute lymphoblastic leukemia, Hodgkin's disease, breast cancer, malignant lymphoma and small cell lung cancer. There is no report about the prevention and treatment effect of vincristine on myocardial fibrosis.

Contents of the invention

Aiming at the above-mentioned deficiencies in the prior art, the present invention provides drugs for effectively preventing or treating myocardial fibrosis, and provides a new application of vincristine. Vincristine can well prevent or treat myocardial fibrosis.

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

A medicine composed of vincristine or its pharmaceutically acceptable salt, ester or solvate, after the vincristine dissolves in the body, the vincristine can effectively prevent or treat myocardial fibrosis.

The above-mentioned myocardial fibrosis generally refers to a kind of heart pathological change in which fibroblasts in myocardial tissue are overactivated, secrete a large amount of collagen fibers and deposit excessively, collagen concentration and collagen volume fraction increase significantly, and various types of collagen are out of balance and arranged in disorder. Caused by chronic inflammation and apoptosis of myocardial tissue.

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

Further, the medicine is a pharmaceutical preparation composed of an effective amount of vincristine and a pharmaceutically acceptable auxiliary material; the auxiliary material is selected from one or more of the following material groups: microcrystalline cellulose, pregelatinized starch , lactose, magnesium stearate, talc, beta cyclodextrin, sodium bicarbonate and croscarmellose sodium.

Further, the dosage of the vincristine: 25ug/kg;

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

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

The beneficial effects of the present invention are:

The present invention develops the therapeutic effect of vincristine on myocardial fibers through in vivo experiments in rats and in vitro cell experiments, and the results prove that vincristine has obvious inhibitory effect on cardiac remodeling; vincristine has obvious therapeutic effect on myocardial fibrosis role and can inhibit myocardial fibrosis.

Description of drawings

Fig. 1 is the chemical structural formula of vincristine of the present invention;

Fig. 2 is the result schematic diagram of the mensuration of cardiac index (Heart index) of the present invention;

Fig. 3 is the result schematic diagram of the degree of myocardial fibrosis of the present invention;

Fig. 4 is the schematic diagram of the expression results of myocardial CollagenI and CollagenIII detected by immunohistochemistry of the present invention;

5 is a schematic diagram of the expression results of myocardial interleukin 1 (IL-1β) and interleukin 18 (IL-18) detected by immunohistochemistry of the present invention;

6 is a schematic diagram of the expression results of IL-1β and IL-18 proteins detected in myocardial tissue by Western blot of the present invention;

Fig. 7 is a schematic diagram of the results of detection of ASC, NLRP3, pro-Caspase-1, and Caspase-1-p20 protein expression levels in myocardial tissue by Western blot of the present invention;

Fig. 8 shows the toxicity of vincristine to cardiac fibroblasts detected by CCK8 of the present invention. Schematic diagram of the results of enzyme-linked immunosorbent assay (Elisa) detection of IL-1β and IL-18 concentrations in the supernatant of cardiac fibroblasts;

Fig. 9 is a schematic diagram of detection results of ASC, NLRP3, pro-Caspase-1, and Caspase-1-p20 protein expression levels in cells detected by Western blot of the present invention.

Detailed ways

Below in conjunction with accompanying drawing 1-9 the present invention will be further described:

Rat in vivo experiments and in vitro cell experiments

Raw materials: 32 8-week-old SPF male SD rats, body weight (200±20g), purchased from the Experimental Animal Center of Guangxi Medical University; vincristine sulfate is a white powder, dissolved in normal saline, purchased from Suleibao Company (2068-78-2), isoproterenol hydrochloride is a white powder, dissolved in normal saline, purchased from U.S. Sigma-Aldrich (I5627). Antibodies used: collagen I (Proteintech, 14695-1-AP), collagen III (Proteintech, 22734-1-AP), NLRP3 (NOVUS, NBP2-12446), ASC (Cell Signaling Technology, 67824), pro-caspase1 ( Abcom, ab179515), cleaved-caspase1-p20 (Proteintech, 22915-1-AP), IL-18 (ab71495; Abcam), IL-1β (31202; Cell Signaling Technology), GAPDH (Proteintech, 60004-1-Ig, 1:5000). Rat interleukin 1β (IL-1β) ELISA kit (China Sangon Bioengineering Company), rat interleukin 18 (IL-18) ELISA kit (China Sangon Bioengineering Company).

Model construction and animal experiments: rats were randomly divided into control group (n=8), myocardial fibrosis model group (n=8) and vincristine low-dose group (25ug/kg, n=8) vincristine high-dose group group (50ug/kg, n=8). Injection of isoproterenol to reproduce myocardial fibrosis model in rats. The vincristine high-dose and low-dose groups were given subcutaneous injections of VCR25ug/kg and 50ug/kg to the rats in the morning, respectively, and the control group and the myocardial fibrosis group were injected with the same amount of normal saline. In the afternoon, the myocardial fibrosis group and the vincristine high-dose and low-dose groups were subcutaneously injected with 5 mg/kg isoproterenol, and the control group was injected with the same amount of normal saline. Eat and drink normally during this period for 10 consecutive days. On the eleventh day, the rats were killed, and various indicators were tested. Remarks: the amount of vincristine added, rats are administered 25ug (low dose group)\50ug (high dose group) of vincristine per kilogram, and vincristine is 824.96 according to the vincristine content in vincristine sulfate. /923.0361 for conversion.

1. Heart weight and cardiac index

After the intervention, the heart weight and body weight of the rats in each group were detected and the heart index (heart weight/body weight) was calculated for comparison. The appearance of panel A in Figure 2 shows that compared with the control group, the heart in the myocardial fibrosis group is more hypertrophic, indicating that the myocardial fiber modeling is successful, while the vincristine high and low dose groups show that the heart volume is smaller than that in the myocardial fibrosis group. The cardiac index (heart mass/rat body weight) of each group rat that shows measurement in the B figure of Fig. 2, model group obviously raises compared with matched group, and the cardiac index value of vincristine high and low dose group is higher than model group decreased, thus confirming that vincristine has an inhibitory effect on cardiac remodeling (*P<0.05).

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

The rat ventricle tissue was fixed in 10% formaldehyde solution, embedded in paraffin and sectioned. Hematoxylin-eosin (HE) staining and Masson staining were performed to observe the tissue changes and the degree of myocardial fibrosis. The fibrous tissue was blue under Masson's staining. The results of HE staining in Figure 3 A are shown in the figure. The cardiomyocytes in the control group are arranged normally, the myocardial fibers are neat, no breaks, and the shape is basically the same; in the myocardial fibrosis group, cardiomyocytes are dissolved, the structure of myocardial fibers is disordered, and inflammatory cells infiltrate ; Myocardial fiber damage and inflammatory cell infiltration were reduced in the vincristine administration group. The results of Masson staining showed that in the normal control group, the red cardiomyocytes were arranged neatly and densely, and a small amount of blue collagen fibers were scattered in the myocardial interstitium; in the myocardial fibrosis group, a large amount of blue collagen deposits were seen in the myocardial interstitium and around blood vessels, and the direction was disordered. Staggered; the vincristine high-dose and low-dose groups had more obvious cardiovascular and interstitial collagen deposition, and the degree of fibrosis was significantly reduced compared with the model group. The ratio of the area of myocardial fibrosis to the total area of rats in each group was calculated by Image J software in Figure 3, B. The myocardial fibrosis group was significantly higher than that in the control group, confirming the success of myocardial fibrosis modeling, while the ratio of the fibrosis area in the vincristine high-dose and low-dose groups was significantly reduced, confirming the therapeutic effect of vincristine on myocardial fibrosis (* P<0.05).

3. Immunohistochemical detection of the expression of CollagenI and CollagenIII in myocardial tissue

Figure 4 A shows that compared with the control group, the expression of CollagenI and CollagenIII in the myocardial interstitium in the myocardial fibrosis group is significantly increased, confirming the successful modeling of myocardial fibrosis. Compared with the myocardial fibrosis group, the expressions of CollagenI and CollagenIII in the vincristine high-dose and low-dose groups decreased. Figure B in Figure 4 is an image analysis using Image J software and similar data statistical results were obtained, further confirming that vincristine can inhibit myocardial fibrosis (*P<0.05).

4. Immunohistochemical detection of the expression of interleukin 1 (IL-1β) and interleukin 18 (IL-18) in myocardial tissue. In panel A of FIG. 5 , compared with the control group, the expressions of IL-1β and IL-18 in the myocardial fibrosis group were significantly increased. Compared with the myocardial fibrosis group, the expressions of IL-1β and IL-18 in the vincristine high-dose and low-dose groups decreased. Image analysis with Image J software also reached the same conclusion, further confirming that vincristine can reduce the expression of IL-1β and IL-18 in myocardial tissue (*P<0.05).

5. Western blotting (Western blot) detection of IL-1β, IL-18 protein expression levels in myocardial tissue. As shown in Figure 6A, compared with the normal group, the expression levels of IL-1β and IL-18 proteins in the myocardial fibrosis model group were significantly increased (*P<0.05). Compared with the model group, the expression levels of IL-1β and IL-18 proteins in vincristine high-dose and low-dose groups were significantly decreased (P<0.05). The same conclusion was obtained by using ImageJ software analysis in Figures B and C of Figure 6, further confirming that vincristine can reduce the expression of IL-1β and IL-18 in myocardial tissue (*P<0.05).

6. Western blotting (Western blot) detection of ASC, NLRP3, pro-Caspase-1, Caspase-1-p20 protein expression levels in myocardial tissue. Figure 7 shows that compared with the normal group, the expression levels of NLRP3 and Caspase-1-p20 proteins in the myocardial fibrosis model group were significantly increased (*P<0.05), confirming the activation of the NLRP3 inflammasome in the heart. Compared with the model group, the expression level of Caspase-1-p20 protein in the vincristine high-dose and low-dose groups was significantly reduced (P<0.05). The expression of , confirmed that vincristine can improve myocardial fibrosis by inhibiting the activation of NLRP3 inflammasome (*P<0.05).

7. Further confirm the inhibitory effect of vincristine on NLRP3 activation through in vitro experiments. Rat cardiac fibroblasts were cultured in primary culture, and the cells were identified by detecting the expression of the fibroblast marker protein Vimentin by immunofluorescence. In Figure 8, A, Vimentin was positively expressed, confirming that the cultured cells were fibroblasts. The toxicity of vincristine to cardiac fibroblasts was studied by Cell CountingKit-8 (CCK8). It is shown in the B diagram of Figure 8 that vincristine with a concentration range of 10-100umol/L has no obvious toxicity to cardiac fibroblasts. Therefore select 10umol/L, 20umol/L, the vincristine of 50umol/L to carry out next experiment. Stimulate fibroblasts with lipopolysaccharide (LPS) + adenosine triphosphate (ATP) to activate NLRP3 inflammasome, add vincristine to explore whether vincristine can inhibit the activation of NLRP3 inflammasome in cardiac fibroblasts, through the enzyme The concentration of IL-1β and IL-18 in the cell supernatant was detected by immunosorbent assay (Elisa). C, D shows that compared with the control group, the concentrations of IL-1β and IL-18 in the cell supernatant of the LPS+ATP stimulation group were significantly increased, confirming the NLRP3 inflammation in fibroblasts after LPS+ATP stimulation body is activated. Compared with LPS+ATP stimulation group, vincristine 10umol/L, 20umol/L, and the concentration of IL-1β in the cell supernatant of 50umol/L group were significantly reduced, proving that vincristine can inhibit cardiogenesis. Activation of NLRP3 inflammasome in fibroblasts (*P<0.05).

8. Western blotting (Western blot) detection of ASC, NLRP3, pro-Caspase-1, Caspase-1-p20 protein expression levels in cells. Figure 9 shows that compared with the normal group, the expression levels of NLRP3 and Caspase-1-p20 proteins in the LPS+ATP stimulation group were significantly increased (*P<0.05), confirming the activation of the NLRP3 inflammasome in fibroblasts. Compared with the LPS+ATP stimulation group, the expression level of Caspase-1-p20 protein in the vincristine 10umol/L, 20umol/L, and 50umol/L groups was significantly reduced (P<0.05), combined with the above-mentioned vincristine decreased The concentration of IL-1β and IL-18 in the cell supernatant confirmed that vincristine can inhibit the activation of NLRP3 inflammasome in cardiac fibroblasts (*P<0.05).

Those skilled in the art will recognize that many variations on the above description are possible, so the examples are merely intended to describe one or more particular implementations.

Although there have been described and described what are considered to be exemplary embodiments of the present invention, it will be apparent to those skilled in the art that various changes and substitutions may 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 inventive central concept described herein. Therefore, the present invention is not limited to the specific embodiments disclosed herein, but the present invention may also include all embodiments and their equivalents falling within the scope of the present invention.

Claims (5)

1. The use of vincristine or a pharmaceutically acceptable salt thereof as the only active ingredient in the preparation of a medicament for treating myocardial fibrosis, characterized in that the pharmaceutically acceptable salt of vincristine is vincristine sulfate. 2. The application according to claim 1, characterized in that, the medicine is an effective dose of vincristine or a pharmaceutically acceptable salt thereof, and a pharmaceutical preparation composed of pharmaceutically acceptable auxiliary materials. 3. application according to claim 2, is characterized in that, described auxiliary material is selected from one or more in the following material groups: microcrystalline cellulose, pregelatinized starch, lactose, magnesium stearate, talcum powder , Beta Cyclodextrin, Sodium Bicarbonate and Croscarmellose Sodium. 4. The application according to claim 2, characterized in that, the dosage form of the pharmaceutical preparation is a liquid or solid dosage form. 5. The application according to claim 2, characterized in that, the route of administration of the pharmaceutical preparation is injection administration.

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