Application of sipeimine in preparation of medicine for treating myocardial fibrosis

Abstract

The invention belongs to the technical field of biology, and particularly discloses application of sipeimine in preparation of a medicine for treating myocardial fibrosis. The invention discloses application of sipeimine in preparation of a medicine for treating myocardial fibrosis, the sipeimine treats myocardial fibrosis by inhibiting combination of cold-induced RNA (Ribonucleic Acid) binding protein and potential transforming growth factor beta binding protein 3 mRNA (Messenger Ribonucleic Acid), and a brand new medicine candidate molecule is provided for prevention and treatment of myocardial fibrosis.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of fritillary alkaloid in the preparation of drugs for treating myocardial fibrosis. Background Technology

[0002] Myocardial fibrosis is a common pathological basis for the progression of many heart diseases to heart failure. It is characterized by excessive activation and proliferation of myocardial fibroblasts, which secrete large amounts of extracellular matrix, leading to myocardial stiffness, impaired diastolic function, and ultimately heart failure. Currently, clinical treatments for myocardial fibrosis are still very limited, mainly relying on angiotensin-converting enzyme inhibitors (ACEIs), angiotensin II receptor blockers (ARBs), and beta-blockers. These drugs primarily delay the fibrosis process indirectly by regulating hemodynamics or the neuroendocrine system. There is still a lack of specific drugs that can directly target and reverse the pathological process of fibrosis, highlighting an urgent clinical need.

[0003] In recent years, research into the pathogenesis of myocardial fibrosis has deepened, revealing that molecules such as cold-induced RNA-binding protein (CIRBP) are upregulated under stress conditions and promote fibroblast phenotypic transformation by regulating related signaling pathways (such as TGF-β / Smad), thus being considered a key link in fibrosis progression. Therefore, identifying bioactive molecules capable of intervening in this pathological process has become an important direction in drug development. Small molecule compounds have attracted considerable attention due to their ease of modification and relatively good drug-like properties; however, highly effective and low-toxicity small molecule inhibitors targeting this specific target or pathway are still relatively scarce, and their specific mechanisms of action and in vivo efficacy require further clarification.

[0004] Sipeimine (Sip) is a steroidal alkaloid compound extracted from the traditional Chinese medicinal herb Fritillaria cirrhosa. Modern pharmacological studies have shown that it possesses various biological activities, including antitussive, expectorant, and anti-inflammatory effects. However, to date, no literature or patents have reported that sipeimine has anti-myocardial fibrosis effects, nor has any research revealed whether it can exert cardioprotective effects by intervening in related targets such as CIRBP. Therefore, exploring new applications of sipeimine in anti-myocardial fibrosis, elucidating its mechanism of action, and developing corresponding drug formulations are of great significance for filling existing treatment gaps and providing new treatment strategies. Summary of the Invention

[0005] This invention aims to provide the application of fritillary alkaloid in the preparation of drugs for treating myocardial fibrosis. This fritillary alkaloid treats myocardial fibrosis by inhibiting the binding of cold-induced RNA-binding protein to potential transforming growth factor β-binding protein 3 mRNA, providing a novel drug candidate molecule for the prevention and treatment of myocardial fibrosis.

[0006] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows: Application of fritillary alkaloids in the preparation of drugs for treating myocardial fibrosis.

[0007] Preferably, the cephalosporin has a structure as shown in formula (I): (I).

[0008] Preferably, the myocardial fibrosis is post-myocardial infarction myocardial fibrosis and / or pressure overload-related myocardial fibrosis.

[0009] Preferably, the cibeneline treats myocardial fibrosis by inhibiting the abnormal activation of myocardial fibroblasts.

[0010] Preferably, the cibeneline treats myocardial fibrosis by inhibiting the binding of cold-induced RNA-binding protein to potential transforming growth factor β-binding protein 3 mRNA.

[0011] The present invention also provides a pharmaceutical composition for treating myocardial fibrosis, comprising the aforementioned cibeneline and a pharmaceutically acceptable carrier.

[0012] Preferably, pharmaceutically acceptable carriers are one or more of the following: nanocarriers, solvents, dispersants, suspending agents, surfactants, isotonic agents, thickeners, preservatives, solid binders, or lubricants.

[0013] The present invention also provides a pharmaceutical formulation comprising a therapeutically effective amount of the aforementioned cibeneine and a pharmaceutically acceptable excipient.

[0014] Preferably, the pharmaceutical preparation includes the following dosage forms: oral preparations (such as tablets, capsules, solutions or suspensions); injectable preparations (such as injectable solutions or suspensions, or injectable dry powders that can be used immediately after being added to water for injection before injection).

[0015] Compared with the prior art, the present invention has the following advantages and technical effects: This invention discloses the application of fritillary bulb alkaloids in the preparation of drugs for treating myocardial fibrosis, revealing for the first time that fritillary bulb alkaloids possess significant anti-myocardial fibrosis activity, providing a novel drug candidate molecule for the prevention and treatment of myocardial fibrosis. In vitro cell experiments confirmed that fritillary bulb alkaloids can effectively inhibit abnormal activation of myocardial fibroblasts induced by CIRBP stimulation, significantly reducing the expression of key fibrosis markers Collagen-I, Vimentin, and α-SMA, thereby blocking the fibrosis process at the cellular level. Further animal experiments showed that under fritillary bulb alkaloid treatment, cardiac function in rats with myocardial infarction was significantly improved, manifested as increased ejection fraction (EF%) and decreased N-terminal pro-B-type natriuretic peptide (NT-proBNP) levels; simultaneously, myocardial tissue pathological staining and protein analysis showed a significant reduction in the degree of myocardial fibrosis and collagen deposition, confirming its clear therapeutic effect at the whole animal level.

[0016] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0017] Figure 1 Images of the rat model in Example 2 are shown. Figure 1 In the image, A is a gross photograph taken after the rat model of myocardial infarction was established. Figure 1 B in the image is a gross photograph of a rat after injecting a drug-loaded hydrogel suspension into the infarcted myocardium. Figure 2 Image showing the identification results of primary cardiac fibroblasts; Figure 3 The effect of cibene alkaloids on the content of Collagen-I, Vimentin and α-SMA in myocardial fibroblasts; Figure 4 This is a quantitative analysis of the effects of cispermine on the levels of Collagen-I, Vimentin, and α-SMA in cardiomyocytes. Figure 4 In this context, A represents the α-SMA result. Figure 4 B in the figure represents the Vimentin result. Figure 4 C in the text represents the Collagen-I result; Figure 5 The results show the effects of fritillary bulb alkaloids on cardiac function in rats, among which... Figure 5 In the figure, A represents the echocardiogram results of the rats at 28 days. Figure 5 B in the graph represents the statistical value of EF% in rats. Figure 5 C in the figure represents the statistical graph of serum NT-proBNP levels; Figure 6 The graph shows the effect of fritillary bulb alkaloids on myocardial tissue proteins. Figure 7HE staining results showing the effect of fritillary alkaloids on myocardial fibrosis. Figure 8 Masson staining results showing the effect of fritillary alkaloids on myocardial fibrosis. Figure 9 The image shows the Sirius red staining results of the effect of cibene alkaloid on myocardial fibrosis. Detailed Implementation

[0018] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0019] Unless otherwise defined, the technical or scientific terms used in this invention shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0020] Source of experimental materials: In this invention, unless otherwise specified, all other test materials and instruments are conventional test materials in the field and can be purchased through commercial channels.

[0021] Example 1 Isolation and identification of primary rat fibroblasts: Enzymatic digestion was used to extract rat cardiac fibroblasts. SPF-grade wild-type SD suckling mice aged 1-3 days were selected (excluding individuals with obvious developmental abnormalities or poor condition). After anesthesia, the anterior chest area was disinfected with alcohol swabs. The sternum was cut open along the left xiphoid process, and the spontaneously beating heart was removed with forceps. Connective tissue and blood clots on the heart were removed (modeling as follows). Figure 1 As shown in the figure, the left and right atria and right ventricle were removed. After washing, digestion, filtration, and centrifugation to collect the precipitate, it was resuspended in a complete culture medium containing 5% fetal bovine serum and 1% penicillin-streptomycin. The resuspended precipitate was then transferred to a sterile six-well cell culture plate and cultured until the cell density reached about 80% for subsequent cell passage.

[0022] Identification of primary fibroblasts: Fibroblast morphology was observed under a microscope, and the results are as follows. Figure 2 As shown.

[0023] Depend on Figure 2 It can be seen that the cells have a flattened structure that is spindle-shaped, fusiform, or star-shaped, and the outline is clear. Secondly, using the cell immunoassay with the aid of a fluorescence microscope, a suitable field of view is randomly selected. Cells with a deeply stained, oval, dense blue nucleus (DAPI) and green filaments in the cytoskeleton (Vimentin) are fibroblasts.

[0024] In vitro cell modeling and treatment methods: The primary myocardial fibroblasts identified above were used in a process of 5 × 10⁻⁶ cells / year. 5The concentration was seeded into 6-well plates and placed in a CO2 incubator. After the cells adhered and reached 80% confluence, the cells were divided into groups: blank control (C), positive control (rC) (cold-induced RNA-binding protein (CIRBP) 60 ng / mL), 50 μM (CIRBP 60 ng / mL + 50 μM cisperidin), and 100 μM (CIRBP 60 ng / mL + 100 μM cisperidin). The cells were then incubated in a CO2 incubator for 48 h.

[0025] Cell protein extraction: Lyse cells on ice for 30 min with 100 μL of RIPA lysis buffer (100:1 ratio of protease inhibitor and phosphatase inhibitor) per well, scrape the cells off and place them in 1.5 mL EP tubes, centrifuge at 4 °C, 12000 rpm for 10 min, and obtain the supernatant as cell protein.

[0026] Determination of Collagen-I and α-SMA content in cellular proteins: After determining the protein concentration using a BCA kit, samples of the same mass were loaded. Using pre-prepared 12-well 4-12% SDS-PAGE gels, the supernatant was used for Western blotting protein separation, ECL chemiluminescence development, and grayscale analysis using ImageJ software. Changes in the protein content of Collagen-I, Vimentin, and α-SMA in cells were measured. The results are shown below. Figure 3 As shown, the quantitative analysis is as follows Figure 4 As shown.

[0027] Depend on Figure 3 and Figure 4 It can be seen that, compared with the control group, the expression of intracellular fibrotic proteins increased after CIRBP stimulation, but the degree of fibrosis was reduced after the addition of cispermine.

[0028] Example 3 Establishment of in vivo animal model: Thirty SD rats (200g-250g) were housed in the animal facility of the Institute of Veterinary Medicine, Lanzhou University for one week to acclimatize to the environment, with free access to water and food. They were randomly divided into three groups: a sham-operated control group (sham), a myocardial infarction model group (MI), and a group receiving cispermine injection after myocardial infarction (MI+Sip, 3mg / kg), with 10 rats in each group.

[0029] In this experiment, the animals underwent left-sided open-chest surgery under general anesthesia and respiratory support. After exposing the heart, the left anterior descending coronary artery was ligated near the lower border of the left atrial appendage to establish an acute myocardial infarction model. Following successful ligation, the myocardium in the ligated area rapidly showed typical changes such as lightening of color and weakened or absent local ventricular wall motion. In the sham surgery group, only sutures were threaded without ligation. Postoperatively, the pleural cavity was closed layer by layer, and routine analgesia and nursing care were provided. On day 28 postoperatively, cardiac function was assessed in each group of rats using small animal echocardiography. On day 28 postoperatively, the animals were sacrificed under deep anesthesia, and myocardial tissue and blood samples were collected for subsequent analysis.

[0030] Intramyocardial injection experiment with fritillaria cirrhosa: After successful ligation of the left anterior descending coronary artery and confirmation of myocardial infarction, 100 μL of fritillaria cirrhosa solution was injected into the infarcted myocardial tissue using a sterile insulin syringe. Routine analgesia and nursing care were provided postoperatively. On postoperative day 28, cardiac function changes were assessed using small animal echocardiography. Animals were sacrificed on postoperative day 28, and myocardial tissue samples were collected to evaluate the degree of myocardial fibrosis and related indicators, as detailed below: Blood sample collection: Rats to be euthanized were anesthetized in a 5% sevoflurane oxygen chamber. The abdominal cavity was opened to expose the abdominal aorta. Blood was collected using disposable blood collection needles and vacuum blood collection tubes. The supernatant was collected by centrifugation and frozen for subsequent detection of serum NT-proBNP levels. Results are as follows: Figure 5 As shown.

[0031] Depend on Figure 5 It was found that the control group (Sham) exhibited regular left ventricular wall motion and large contraction amplitude; while the myocardial infarction model group (MI) showed significantly weakened left ventricular wall motion, indicating significant impairment of left ventricular systolic function after myocardial infarction. Compared with the model group, the MI+Sip group showed increased left ventricular wall motion amplitude and improved contraction morphology after myocardial infarction. Further quantitative analysis showed that the ejection fraction (EF) in the MI group was significantly lower than that in the control group; the EF in the MI+Sip group was significantly higher than that in the model group. Figure 5 (Middle B). Meanwhile, serum NT-proBNP levels in the MI group were significantly higher than those in the Sham group; NT-proBNP levels in the MI+Sip group were significantly lower than those in the model group. Figure 5 (C). The above results indicate that the fritillary alkaloids described in this invention can improve left ventricular systolic function and reduce heart failure-related biochemical indicators in a myocardial infarction model, thereby having a clear ameliorative effect on cardiac function impairment after myocardial infarction.

[0032] Myocardial tissue specimen collection: After exsanguination via the abdominal aorta, the heart of the rat was quickly removed, rinsed with pre-cooled physiological saline to remove blood contamination, and excess water was absorbed with gauze. Myocardial tissue from the infarcted area was fixed in 4% paraformaldehyde for 48 hours, followed by routine paraffin embedding, sectioning, HE staining, Masson and Sirius red staining, and microscopic observation of myocardial morphology. The remaining myocardial tissue was wrapped in aluminum foil and rapidly frozen in liquid nitrogen for long-term storage at -80°C for subsequent parameter determination.

[0033] Determination of Collagen-I, Vimentin, and α-SMA content in myocardial tissue: An appropriate amount of myocardial tissue was added to RIPA lysis buffer and homogenized at low temperature. The tissue was centrifuged at 12000g at 4℃ for 3 min. The supernatant was collected, and sample buffer was added and boiled at 100℃ for 10 min to obtain protein samples for subsequent determination. The results are as follows: Figures 6-9 As shown.

[0034] Using pre-prepared 12-well 4-12% SDS-PAGE gels, the above protein samples were subjected to Western blotting for protein separation, ECL luminescence development, and grayscale analysis was performed using ImageJ software to determine the changes in the content of Collagen-I and α-SMA in cardiac tissue.

[0035] Depend on Figure 6 It was found that after intramyocardial injection of cispermine hydrogel suspension, the levels of fibrosis-related proteins α-SMA and Collagen-I in rat myocardial tissue decreased, effectively reducing the progression of myocardial fibrosis.

[0036] Depend on Figure 7 It was found that the myocardial tissue structure in the Sham group was intact, with regular and continuous arrangement of myocardial fibers, and no obvious necrotic foci or abnormal inflammatory infiltration were observed. In the MI group, significant damage to the myocardial tissue structure was observed, with infarct / damage areas, broken and disordered arrangement of myocardial fibers, local tissue loosening, and pathological changes such as inflammatory cell infiltration. Compared with the MI group, the degree of myocardial tissue structural damage was reduced in the MI+Sip group, the continuity and arrangement of myocardial fibers were improved, and inflammatory infiltration and tissue edema / loosening were reduced, suggesting that the formulation of this invention can alleviate histological damage after myocardial infarction.

[0037] Depend on Figure 8 It was observed that the myocardial tissue in the Sham group was mainly composed of muscle fibers, with no significant blue-stained collagen fiber deposition. In the MI group, a large amount of blue-stained collagen fiber deposition was observed in and around the infarct area, distributed in sheet-like / band-like patterns, indicating a significant increase in collagen deposition and a marked fibrotic reaction. Compared with the MI group, the MI+Sip group showed a significant reduction in blue-stained collagen deposition and a decrease in the extent and degree of fibrosis, indicating that the formulation of this invention can effectively inhibit excessive collagen deposition after myocardial infarction and improve fibrotic remodeling.

[0038] Depend on Figure 9 It was observed that only a small amount of physiological collagen was distributed in the Sham group, with weak red staining signal and a small area. In the MI group, red staining collagen in and around the infarct area was significantly enhanced and widely distributed, indicating a significant increase in collagen deposition and scar formation. Compared with the MI group, the red staining collagen signal in the MI+Sip group was significantly weakened, and the collagen deposition area was reduced, indicating that the formulation of this invention can reduce the level of collagen deposition after infarction, alleviate the process of scar fibrosis, and thus improve myocardial tissue remodeling.

[0039] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. Application of fritillary alkaloids in the preparation of drugs for the treatment of myocardial fibrosis.

2. The application according to claim 1, characterized in that, The cisternine has a structure as shown in formula (I): （I）。 3. The application according to claim 1 or 2, characterized in that, The myocardial fibrosis refers to post-myocardial infarction myocardial fibrosis and / or pressure overload-related myocardial fibrosis.

4. The application according to claim 3, characterized in that, The cephalomyelin mentioned above treats myocardial fibrosis by inhibiting the abnormal activation of myocardial fibroblasts.

5. The application according to claim 3, characterized in that, The cibene alkaloid treats myocardial fibrosis by inhibiting the binding of cold-induced RNA-binding protein to potential transforming growth factor β-binding protein 3 mRNA.

6. A pharmaceutical composition for treating myocardial fibrosis, characterized in that, It comprises the fritillary alkaloid as described in claim 1 and a pharmaceutically acceptable carrier.

7. The pharmaceutical composition according to claim 6, characterized in that, Pharmaceutically acceptable carriers are one or more of the following: nanocarriers, solvents, dispersants, suspending agents, surfactants, isotonic agents, thickeners, preservatives, solid binders, or lubricants.

8. A pharmaceutical preparation, characterized in that, The product comprises a therapeutically effective amount of the cephalosporin as described in claim 1, and a pharmaceutically acceptable excipient.

Images