CN116617360A - Application of radix pseudostellariae cyclic peptide B in preparation of drugs for improving cardiac function - Google Patents
Application of radix pseudostellariae cyclic peptide B in preparation of drugs for improving cardiac function Download PDFInfo
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
- A61K38/12—Cyclic peptides, e.g. bacitracins; Polymyxins; Gramicidins S, C; Tyrocidins A, B or C
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
- A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Veterinary Medicine (AREA)
- Diabetes (AREA)
- Chemical & Material Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Engineering & Computer Science (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Public Health (AREA)
- General Health & Medical Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- General Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Immunology (AREA)
- Heart & Thoracic Surgery (AREA)
- Cardiology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Epidemiology (AREA)
- Medicines Containing Plant Substances (AREA)
- Emergency Medicine (AREA)
- Endocrinology (AREA)
- Hematology (AREA)
- Obesity (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Gastroenterology & Hepatology (AREA)
Abstract
The invention belongs to the technical field of medicines, and provides an application of pseudostellaria root cyclic peptide B in preparing a medicine for improving cardiac function. The radix pseudostellariae cyclopeptide B can inhibit collagen formation of myocardial tissues, maintain mitochondrial homeostasis of myocardial cells, maintain mitochondrial autophagy homeostasis of myocardial cells and inhibit apoptosis of myocardial cells. The invention discovers that the pseudostellate cyclopeptide B is combined with MAVS, so that the aggregation activation of MAVS is activated, and the mitochondrial autophagy signal is induced, thereby mediating the turnover of damaged mitochondria, promoting the update of the mitochondria, and providing a new theoretical basis and a new drug treatment target for the treatment of diabetic cardiomyopathy.
Description
Technical Field
The invention belongs to the technical field of medicines, and particularly relates to application of pseudostellaria root cyclic peptide B in preparation of a medicine for improving cardiac function.
Background
As a metabolic disease characterized by hyperglycemia, diabetes can cause systemic multi-organ, multi-system damage, where the risk factors for diabetic cardiomyopathy (Diabetic cardiomyopathy, DCM) predominate, leading to death from heart failure in the patient. Diabetic cardiomyopathy is a heart disease caused by blood sugar and/or hyperlipidemia, etc., and heart failure is a major clinical manifestation of diabetic cardiomyopathy. The onset of diabetic cardiomyopathy is suppressed, the earliest manifestations are usually dominated by impaired diastolic function, and progress to overt diastolic heart failure in the later stages, with or without ventricular enlargement, which is usually accompanied by reduced systolic function.
At present, hypoglycemic agents, angiotensin converting enzyme inhibitors, angiotensin II antagonists, beta receptor blockers and the like are adopted for clinical treatment, and although the hypoglycemic agents control blood sugar concentration, the hypoglycemic agents have insignificant effects on heart, cardiovascular drugs have the risk of aggravating hyperglycemia, so the curative effect is limited. The main reason is that the specific pathogenesis of the diabetic cardiomyopathy is unknown, and no specific treatment method exists.
Chinese patent application 202110697230.8 discloses a new use of iCRT14, in particular to a new use of iCRT14 in preparing a medicament for treating type 2 diabetic cardiomyopathy. The iCRT14 is capable of lowering blood glucose in type 2 diabetic cardiomyopathy, improving myocardial remodeling in type 2 diabetic cardiomyopathy, improving systolic and diastolic dysfunction caused by type 2 diabetic cardiomyopathy, and inhibiting high-glucose high-insulin-induced cardiomyocyte hypertrophy.
Chinese patent application 201810023779.7 discloses the application of tumor necrosis factor alpha-inducing protein 3 in preparing medicine for treating diabetic cardiomyopathy. Establishing a mouse diabetic cardiomyopathy model through High Fat Diet (HFD) induction, infecting primary rat cardiomyocytes through constructing a recombinant adenovirus vector (AdA) which overexpresses A20, and establishing an in vitro cell model of the diabetic cardiomyopathy by high-level fatty acid stimulation, and simultaneously establishing a mouse diabetic cardiomyopathy model through HFD induction by taking WT mice and heart-specific A20 gene knockout mice (A20 CKO) as experimental subjects, wherein the protein expression level of A20 in heart tissues of mice of HFD 24W is found to be significantly reduced.
None of the drugs provided in the above application is related to a specific therapeutic target, so revealing the pathogenesis of diabetic cardiomyopathy, defining the therapeutic target and finding a drug effective in treatment is a problem to be solved in the art.
The radix pseudostellariae cyclic peptide B is one of main components of traditional Chinese medicine radix pseudostellariae, and is one of the current radix pseudostellariae cyclic peptide components which is more studied. Modern researches have shown that radix Pseudostellariae mainly contains chemical components such as saccharides, cyclic peptides, saponins, amino acids, volatile oil, trace elements, etc. Modern pharmacological researches show that the traditional Chinese medicine composition has the effects of enhancing immunity, resisting bacteria, resisting lipid peroxidation/inflammation and the like, and has few reports on the effects of the traditional Chinese medicine composition on diabetic cardiomyopathy.
Disclosure of Invention
In order to solve the technical problems, the invention explores the pathogenesis of the diabetic cardiomyopathy, defines a treatment target point, and provides the application of the pseudostellaria root cyclic peptide B in medicines for improving cardiac function and preventing and/or treating the cardiomyopathy.
The technical scheme provided by the invention is as follows:
the first aspect of the invention provides an application of pseudostellaria root cyclic peptide B in preparing a medicine for improving cardiac function.
In some embodiments, the agent is an agent that binds to a mitochondrial antiviral signaling protein.
In some embodiments, the agent is an agent that activates mitochondrial antiviral signaling proteins.
In some embodiments, the drug improves cardiac function by one or more of the following mechanisms:
(1) Inhibiting collagen formation in myocardial tissue;
(2) Maintaining mitochondrial homeostasis of cardiomyocytes;
(3) Maintaining mitochondrial autophagy homeostasis of cardiomyocytes;
(4) Inhibit apoptosis of cardiomyocytes.
In some embodiments, the drug comprises a small molecule substance, e.g., a small molecule chemical such as a small molecule compound, or a small molecule biological such as a small molecule active peptide-conjugate;
and/or the drug comprises a macromolecular substance, for example a macromolecular biological substance such as an antibody-conjugate, or a macromolecular chemical substance such as a macromolecular compound;
and/or the medicament comprises an animal and/or plant extract.
In some embodiments, the small molecule compound comprises a pharmaceutically acceptable salt, a solvate of a compound, a solvate of a pharmaceutically acceptable salt of a compound, or a crystalline form of a compound.
In some embodiments, the medicament comprises an injectable formulation and/or an oral formulation.
The second aspect of the invention provides an application of pseudostellaria root cyclic peptide B in preparing a medicament for preventing and/or treating cardiomyopathy.
In some embodiments, the cardiomyopathy is diabetic cardiomyopathy.
In some embodiments, the agent is an agent that binds to a mitochondrial antiviral signaling protein.
In some embodiments, the agent is an agent that activates mitochondrial antiviral signaling proteins.
In some embodiments, the medicament prevents and/or treats cardiomyopathy by one or more of the following mechanisms:
(1) Inhibiting collagen formation in myocardial tissue;
(2) Maintaining mitochondrial homeostasis of cardiomyocytes;
(3) Maintaining mitochondrial autophagy homeostasis of cardiomyocytes;
(4) Inhibit apoptosis of cardiomyocytes.
In some embodiments, the drug comprises a small molecule substance, e.g., a small molecule chemical such as a small molecule compound, or a small molecule biological such as a small molecule active peptide-conjugate;
and/or the drug comprises a macromolecular substance, for example a macromolecular biological substance such as an antibody-conjugate, or a macromolecular chemical substance such as a macromolecular compound;
and/or the medicament comprises an animal and/or plant extract.
In some embodiments, the small molecule compound comprises a pharmaceutically acceptable salt, a solvate of a compound, a solvate of a pharmaceutically acceptable salt of a compound, or a crystalline form of a compound.
In some embodiments, the medicament comprises an injectable formulation and/or an oral formulation.
The third aspect of the invention provides the use of a pharmaceutical formulation for the preparation of a medicament for improving cardiac function, characterized in that it comprises pseudostellaria root cyclic peptide B, or a pharmaceutically acceptable salt thereof, a solvate of a compound, a solvate of a pharmaceutically acceptable salt of a compound, or a crystalline form of a compound, and at least one pharmaceutically acceptable carrier or excipient.
In some embodiments, the pseudostellaria root cyclic peptide B is provided in the form of a monomer or in the form of an animal and/or plant extract comprising the same.
In some embodiments, the pharmaceutically acceptable carrier or excipient is selected from at least one of solvents, diluents, disintegrants, precipitation inhibitors, surfactants, glidants, binders, lubricants, dispersants, suspending agents, isotonic agents, thickening agents, emulsifiers, preservatives, stabilizers, hydration agents, emulsifying accelerators, buffers, absorbents, colorants, flavoring agents, sweeteners, ion exchangers, mold release agents, coating agents, flavoring agents, and antioxidants.
In some specific embodiments, the formulation is an injectable formulation and/or an oral formulation.
In a fourth aspect, the present invention provides the use of a pharmaceutical formulation for the manufacture of a medicament for the prophylaxis and/or treatment of cardiomyopathy.
In some embodiments, the pseudostellaria root cyclic peptide B is provided in the form of a monomer or in the form of an animal and/or plant extract comprising the same.
In some embodiments, the pharmaceutically acceptable carrier or excipient is selected from at least one of solvents, diluents, disintegrants, precipitation inhibitors, surfactants, glidants, binders, lubricants, dispersants, suspending agents, isotonic agents, thickening agents, emulsifiers, preservatives, stabilizers, hydration agents, emulsifying accelerators, buffers, absorbents, colorants, flavoring agents, sweeteners, ion exchangers, mold release agents, coating agents, flavoring agents, and antioxidants.
In some specific embodiments, the formulation is an injectable formulation and/or an oral formulation.
The invention provides an application of pseudostellaria root cyclic peptide B in medicines for improving cardiac function and preventing and/or treating cardiomyopathy. The radix pseudostellariae cyclopeptide B can inhibit collagen formation of myocardial tissues, maintain mitochondrial homeostasis of myocardial cells, maintain mitochondrial autophagy homeostasis of myocardial cells and inhibit apoptosis of myocardial cells. The invention discovers that the pseudostellate cyclopeptide B is combined with MAVS, so that the aggregation activation of MAVS is activated, and the mitochondrial autophagy signal is induced, thereby mediating the turnover of damaged mitochondria, promoting the update of the mitochondria, and providing a new theoretical basis and a new drug treatment target for the treatment of diabetic cardiomyopathy.
Drawings
FIG. 1 is a cardiac ultrasound of mice from the normal (control), model and radix Pseudostellariae (Pseudostellaria cyclopeptide B) groups.
Fig. 2 is a statistical plot of cardiac ejection fraction (EF%) of mice from the normal (control), model and pseudostellaria (pseudostellaria cyclopeptide B) groups.
Fig. 3 is a graph showing the blood glucose statistics of mice from the normal (control), model and radix Pseudostellariae (pseudostellaria cyclopeptide B) groups.
FIG. 4 is a chart showing the fluorescence staining of collagen Masson in myocardial tissue of mice from the normal (control), model and radix Pseudostellariae (Pseudostellaria cyclopeptide B) groups.
FIG. 5 is a graph showing the results of transmission electron microscopy of myocardial tissue of mice in the normal (control), model and radix Pseudostellariae (Pseudostellaria cyclopeptide B) groups.
FIG. 6 is a graph showing Western Blot results of mitochondrial autophagy-related proteins from mouse myocardial tissue of the normal (control), model and radix Pseudostellariae (Pseudostellate cyclopeptide B) groups.
In fig. 7, a is a normal (control) group, B is a model group, C is a flow cell scattergram of the apoptosis rate of the rat cardiomyocyte line (H9C 2) of the pseudostellaria root (pseudostellaria root cyclic peptide B) group.
FIG. 8 is a Mitosox fluorescent staining pattern of rat cardiomyocyte lines (H9 c 2) of the normal (control), model and pseudostellaria (pseudostellaria cyclopeptide B) groups.
FIG. 9 is a statistical plot of Mitosox fluorescence staining of rat cardiomyocyte lines (H9 c 2) from the normal (control), model and radix Pseudostellariae (Pseudostellaria cyclopeptide B) groups.
FIG. 10 is a JC-1 fluorescent staining pattern of rat cardiomyocyte lines (H9 c 2) of the normal (control), model and radix Pseudostellariae (Pseudostellaria cyclopeptide B) group.
FIG. 11 is a fluorescent staining statistical chart of J-monomers (J-monomers) of rat cardiomyocyte lines (H9 c 2) of the normal (control), model and pseudostellaria root (pseudostellaria root-cyclopeptide B) group.
FIG. 12 is a fluorescent staining statistical chart of J-aggregates (J-aggregates) of rat cardiomyocyte lines (H9 c 2) of the normal (control), model and pseudostellaria root (pseudostellaria root-cyclopeptide B) groups.
Detailed Description
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly used in the art to which this invention belongs. For the purposes of explaining the present specification, the following definitions will apply, and terms used in the singular will also include the plural and vice versa, as appropriate.
The terms "a" and "an" as used herein include plural referents unless the context clearly dictates otherwise. For example, reference to "a cell" includes a plurality of such cells, equivalents thereof known to those skilled in the art, and so forth.
The diabetic cardiomyopathy belongs to the categories of diabetes, palpitation, chest stuffiness, and true heart pain in the traditional Chinese medicine. The pathogenesis of the diabetic cardiomyopathy is complex and is often summarized as deficiency, phlegm and blood stasis, which are marked by deficiency, deficiency of both qi and yin and accumulation of phlegm and blood stasis, so that the symptoms are treated by tonifying qi, nourishing yin, reducing phlegm and removing blood stasis. Because qi can produce blood and promote the production of body fluid, "qi can promote blood circulation", "qi can promote water circulation"; qi excess results in smooth qi movement, and phlegm and blood stasis are removed automatically, so it is mainly indicated for qi-tonifying.
The radix pseudostellariae is a dry root tuber of Pax ex Pax et Hoffm in the new from the herbal, namely the child ginseng Pseudostellaria heterophylla (Miq.) of the Caryophyllaceae, is sweet and slightly bitter, and has the effects of tonifying qi, strengthening spleen, promoting the production of body fluid and moistening lung, and is a common medicament for treating diabetic cardiomyopathy in traditional Chinese medicine. Modern researches have shown that radix Pseudostellariae mainly contains chemical components such as saccharides, cyclic peptides, saponins, amino acids, volatile oil, trace elements, etc. Modern pharmacological researches have shown that it has the functions of enhancing immunity, resisting bacteria, resisting lipid peroxidation/inflammation, reducing blood sugar and lipid, resisting myocardial infarction/heart failure, etc.
The pseudostellaria root cyclic peptide B is taken as a main drug effect substance of the pseudostellaria root, is hardly singly used for treating diseases in the prior research, and has not been studied for the effect of the pseudostellaria root cyclic peptide B on diabetic cardiomyopathy. The invention obviously improves the heart function of animals and reduces blood sugar by taking the pseudostellaria root cyclic peptide B for treatment under the diabetic cardiomyopathy model. The invention discloses important effects of pseudostellaria root cyclic peptide B in remarkably inhibiting myocardial collagen formation, maintaining integrity of mitochondria and the like under a mouse diabetic cardiomyopathy model, and no related report exists in the world at present, so that the pseudostellaria root cyclic peptide B provided by the invention is expected to provide a new theoretical basis and a new drug treatment target for the subsequent treatment of diabetic cardiomyopathy.
The radix pseudostellariae cyclic peptide B is [ ring- (glycine-leucine-proline-isoleucine-phenylalanine) ], and the amino acid sequence of the radix pseudostellariae cyclic peptide B is shown as SEQ ID NO:1 is shown as follows: GGLPPPIF.
The present invention will be described in further detail with reference to the following examples in order to make the objects, technical solutions and advantages of the present invention more apparent. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. All reagents or equipment were commercially available as conventional products without the manufacturer's attention. Numerous specific details are set forth in the following description in order to provide a better understanding of the invention. The specific embodiments described herein are for purposes of illustration only and are not to be construed as limiting the invention in any way. In addition, in the following description, descriptions of well-known structures and techniques are omitted so as not to unnecessarily obscure the present invention. Such structures and techniques are also described in a number of publications.
The material instrument used in the invention comprises:
experimental animals: male mice C57BL/6 (Shanghai Laike laboratory animal Limited), were kept in the Zhejiang university animal center SPF environment.
The instrument and reagents are shown in table 1:
TABLE 1 instruments and reagents used in the invention
| Reagents/apparatus | Branding/suppliers | Goods number/model |
| Mouse heart ultrasonic instrument | Visualsonics | vevo2100 |
| Streptozotocin | Merck (Merck) | 242-646-8 |
| Radix pseudostellariae cyclic peptide B | Chengdu Egya Biotech Co.Ltd | AF22040601 |
| Pentobarbital | Makeup reagent | M008574 |
| Masson dye liquor | Soy pal | G1340 |
| CD31 | Abcam | ab28364 |
| SMA dye liquor | Abcam | ab240678 |
| TUNEL kit | Biyun Tian (a kind of Chinese character) | C1089 |
| Transmission electron microscope | LVEM 5 | / |
| MAVS | CST | 83000S |
| Bcl2L13 | Abcam | ab203516 |
| FUNDC1 | CST | 49240S |
| MUL1 | Abcam | Ab155511 |
| BNIP3L | CST | 12396S |
| Prohibitin 2 | CST | Ab154589 |
| H9C2 cell line | ATCC | BFN60804388 |
| JC-1 fluorescent probe | Biyun Tian (a kind of Chinese character) | C2006 |
| MitoSox staining kit | Siemens fly | M36008 |
EXAMPLE 1 Effect of Pseudostellaria root cyclic peptide B on cardiac function and blood sugar in diabetic cardiomyopathy mice
1) Preparation of diabetic cardiomyopathy mouse model
Building a diabetic cardiomyopathy mouse animal model by adopting Streptozotocin (STZ) modeling: the mice diabetes model was prepared by intraperitoneal injection of STZ. Mice were fasted for 8 hours per day and were intraperitoneally injected with STZ 55mg/kg (mass ratio of STZ to mice, where STZ is in mg and mice are in kg) at a volume of 200 μl per 20g of mice. Mice with blood glucose levels exceeding 16.5mM after 1 week were determined to be successfully modeled after 5 consecutive days, with blood glucose testing using a ROCHE glucometer.
2) Grouping and administration
Normal control, model, pseudostellaria root cyclic peptide B dosing groups of 10 animals each. Administration was started 4 weeks after STZ injection, and index detection was performed 4 weeks after continuous administration.
3) Index detection
(1) Body weight and blood glucose monitoring
Mice were tested for body weight every 2 weeks and blood glucose levels were tested using a ROCHE glucometer.
(2) Cardiac function detection
The animal blind method adopts an echocardiogram to observe the end inner diameters of the left ventricle in the systolic and diastolic phases and the heart ejection fraction; hemodynamic measurement: mice were anesthetized and immobilized, the right common carotid artery was isolated, pressure sensor connected and arterial pressure recorded. The left ventricular systolic pressure, the left ventricular end diastolic pressure, the maximum rate of rise in the left indoor pressure, and the maximum rate of fall in the left indoor pressure are recorded, respectively.
4) Statistical treatment
Data processing analysis is carried out by using GraphPad software, the results are all expressed by mean ± variance, the comparison between groups adopts single factor variance analysis, and P < 0.05 indicates that the difference has statistical significance.
5) Experimental results
The results show that STZ-induced cardiac dysfunction in diabetic cardiomyopathy mice was improved after treatment with pseudostellaria root cyclic peptide B (fig. 1); wherein the EF value is reduced by 40% after STZ induction, and the EF value is increased by 30% after administration of pseudostellaria root cyclic peptide B (figure 2); the treatment with pseudostellaria root cyclic peptide B was not apparent on glycemic control (fig. 3).
EXAMPLE 2 Effect of Pseudostellaria root cyclopeptide B on collagen formation and mitochondria in myocardial tissue of diabetic cardiomyopathy mice
1) Preparation of diabetic cardiomyopathy mouse model
As in example 1.
2) Grouping and administration
Normal control, model, pseudostellaria root cyclic peptide B dosing groups of 10 animals each. Administration was started 4 weeks after STZ injection, and index detection was performed 4 weeks after continuous administration.
3) Detection index
Taking a mouse heart, flushing the heart with physiological saline, embedding a heart sample OCT (optimal cutting temperature) embedding medium, and detecting pathology related indexes after frozen sections: masson staining, inverted microscope observation of collagen formation, CD31 and SMA staining to detect angiogenesis and TUNEL staining to detect myocardial apoptosis. And detecting myocardial mitochondrial autophagy and morphology by a transmission electron microscope.
4) Image processing
Data processing analysis was performed using ImageJ software.
5) Experimental results
The results show that myocardial tissue collagen staining shows that myocardial tissue of diabetic cardiomyopathy mice has a large amount of collagen production; pseudostellaria root cyclic peptide B significantly inhibited collagen formation after administration (fig. 4). Meanwhile, the transmission electron microscope result shows that partial sarcomere structure of the diabetic cardiomyopathy mice is destroyed, myofilaments are broken and dissolved, mitochondria are arranged in disorder and swell, and most of cristae is broken; the pseudostellaria root cyclic peptide B can repair the sarcomere structure and restore the mitochondrial morphology after being administered (figure 5).
Example 3 Effect of Pseudostellaria root cyclopeptide B on the autophagy-related proteins of mouse myocardium in diabetic cardiomyopathy
1) Preparation of diabetic cardiomyopathy mouse model
As in example 1.
2) Grouping and administration
As in example 2.
3) Detection index
Western Blot method for detecting myocardial tissue mitochondrial autophagy related protein expression: parkin, MAVS, bcl2L13, FUNDC1, MUL1, BNIP3L and Prohibitin 2.
4) Statistical treatment
Data processing analysis was performed using ImageJ software.
5) Experimental results
Mitochondrial antiviral signaling protein (Mitochondrial antiviral-signaling protein, MAVS) is a key protein that has been recently found to localize to the outer mitochondrial membrane, promoting mitochondrial autophagy to exert mitochondrial homeostasis.
The results showed that Parkin was significantly reduced in diabetic myocardium to induce loss of mitochondrial autophagy (fig. 6). Mitochondrial autophagy may also be mediated by mitochondrial receptor proteins: prohibiin 2, bcl2-L-13, FUNDC1, MUL1, BNIP3L and MAVS. Therefore, the inventors extracted myocardial tissue proteins, and studied the above mitochondrial autophagy-related proteins, and found that STZ-induced protein expression of Parkin and MAVS was significantly reduced in diabetic cardiomyopathy mice, while there was no effect on the profhibiin 2, bcl2-L-13, fusion 1, mul1 and BNIP3L proteins, suggesting that mitochondrial autophagy loss was related to Parkin and MAVS in STZ-induced diabetic cardiomyopathy (fig. 6). The protein expression of MAVS was significantly up-regulated after treatment with pseudostellate cyclopeptide B, with no effect on Parkin (FIG. 6), suggesting that mitochondrial autophagy modulation of pseudostellate cyclopeptide B is associated with only the MAVS pathway. The invention discovers that the radix pseudostellariae cyclic peptide B is used as a substrate and plays a role by combining with MAVS.
Example 4 Effect of Pseudostellaria root cyclic peptide B on the high sugar-induced myocardial apoptosis Rate
1) Preparation of high sugar injury H9c2 cell model
After treatment of the in vitro cultured rat cardiomyocyte cell line (H9 c 2) with a (100 μm concentration) glucose solution (24 hours), a high sugar damage H9c2 cell model was obtained.
2) Grouping and administration
Dividing the H9c2 cells into a normal control group, a model group and a radix pseudostellariae cyclic peptide B administration group, and administering glucose solutions with the same volume concentration into the model group and the administration group, wherein the radix pseudostellariae cyclic peptide B is additionally added into the administration group.
3) Detection index
After cell plating grouping treatment, the Annexin V-FITC apoptosis detection kit (Annexin V-FITC Apoptosis Detection Kit) marks phosphatidylserine on the surface of apoptotic cell membranes and reflects the apoptosis rate of cells.
Annexin is a class of calcium ion-dependent phospholipid-binding proteins widely distributed in eukaryotic cell cytoplasm and involved in intracellular signal transduction. However, only Annexin V has been reported to modulate some PKC activities.
Annexin V selectively binds to Phosphatidylserine (PS). Phosphatidylserine is predominantly distributed on the inner side of the cell membrane, i.e. the side adjacent to the cell plasma. In the early stages of apoptosis, different types of cells can evert phosphatidylserine to the cell surface, i.e., outside the cell membrane. Phosphatidylserine promotes coagulation and inflammatory reactions upon exposure to the cell surface. And Annexin V can block procoagulant and pro-inflammatory activities of phosphatidylserine after binding to phosphatidylserine everting to the cell surface.
The important feature of phosphotidyl serine, valgus apoptosis, can be detected very simply and directly by flow cytometry or fluorescence microscopy using a fluorescent probe FITC labeled Annexin V, i.e., annexin V-FITC, with green fluorescence.
5) Experimental results
The results showed that H9c2 cardiomyocyte staining showed an increase in apoptosis rate of about 40% in the diabetic cardiomyocyte model and a decrease in cardiomyocyte apoptosis rate of about 20% after treatment with pseudostellate cyclopeptide B (fig. 7).
Example 5 Effect of Pseudostellaria root cyclic peptide B on high glucose-induced mitochondrial membrane potential and active oxygen in cardiomyocytes
1) Preparation of high sugar injury H9c2 cell model
After treatment of in vitro cultured H9c2 cells (24 hours) with (100 μm concentration) glucose solution, a high sugar damage H9c2 cell model was obtained.
2) Grouping and administration
Dividing the H9c2 cells into a normal control group, a model group and a radix pseudostellariae cyclic peptide B administration group, and administering glucose solutions with the same volume concentration into the model group and the administration group, wherein the radix pseudostellariae cyclic peptide B is additionally added into the administration group.
3) Detection index
After cell plating grouping treatment, JC-1 fluorescent probe marks the mitochondrial membrane potential of living cells and reflects the opening degree of the mPTP pore channel; mitoSox staining detects mitochondrial endogenous ROS.
The Mitosox Red superoxide indicator is a novel fluorescent dye and specifically targets mitochondria in living cells. Oxidation of the MitoSOX reagent with mitochondrial superoxide produces bright green or red fluorescence. Mitochondrial production superoxide can be visualized by fluorescence microscopy using a MitoSOX superoxide indicator. The indicator permeates living cells and selectively targets mitochondria therein. These indicators are rapidly oxidized by superoxide but not by other Reactive Oxygen Species (ROS) and Reactive Nitrogen Species (RNS). The oxidation products fluoresce strongly.
JC-1 is an ideal fluorescent probe widely used for detecting mitochondrial membrane potential, and can detect the mitochondrial membrane potential of cells, tissues or purification. At higher mitochondrial membrane potentials, JC-1 aggregates in the matrix of mitochondria (matrix), forming polymers (J-aggregates), which can produce red fluorescence; at low mitochondrial membrane potential, JC-1 cannot aggregate in the matrix of mitochondria, and JC-1 is a monomer (monomer) and can generate green fluorescence. This allows for a very convenient detection of changes in mitochondrial membrane potential by fluorescence color transitions. The relative proportion of red-green fluorescence is often used to measure the proportion of mitochondrial depolarization. The drop in mitochondrial membrane potential is a hallmark event in the early phase of apoptosis. The decrease in cell membrane potential can be readily detected by the transition of JC-1 from red to green fluorescence, and the transition of JC-1 from red to green fluorescence can also be used as a detection indicator for early apoptosis.
4) Statistical treatment
Data processing analysis was performed using ImageJ, graphPad software, the results were all expressed as mean ± variance, the comparison between groups was performed using one-way variance analysis, and P < 0.05 indicated that the differences were statistically significant.
5) Experimental results
The results showed that H9c2 cardiomyocyte staining showed an increase in ROS content in the diabetic cardiomyocyte model resulting in apoptosis (fig. 8), and that the ROS content in the cardiomyocytes was reduced by about 20% after administration of pseudostellate cyclopeptide B (fig. 9). Statistical results showed significant differences between the different groups.
Cardiomyocyte lineage staining showed a decrease in mitochondrial membrane potential in the diabetic cardiomyocyte model leading to apoptosis (fig. 10); the pseudostellaria root cyclic peptide B can obviously improve the mitochondrial membrane potential of myocardial cells after being administered. Statistical results showed that the model group membrane potential was reduced by about 20% compared to the normal group, while the radix pseudostellariae treatment group increased by about 10% compared to the model group membrane potential. (FIGS. 11 and 12).
Claims (9)
1. Application of radix Pseudostellariae cyclic peptide B in preparing medicine for improving cardiac function is provided.
2. The use according to claim 1, wherein the medicament is a medicament that binds mitochondrial antiviral signaling proteins;
preferably, the drug is a drug that activates mitochondrial antiviral signaling proteins;
preferably, the medicament improves cardiac function by one or more of the following mechanisms:
(1) Inhibiting collagen formation in myocardial tissue;
(2) Maintaining mitochondrial homeostasis of cardiomyocytes;
(3) Maintaining mitochondrial autophagy homeostasis of cardiomyocytes;
(4) Inhibit apoptosis of cardiomyocytes.
3. The use according to claim 1 or 2, wherein the medicament comprises a small molecule substance, e.g. a small molecule chemical substance such as a small molecule compound, or a small molecule biological substance such as a small molecule active peptide-conjugate;
and/or the drug comprises a macromolecular substance, for example a macromolecular biological substance such as an antibody-conjugate, or a macromolecular chemical substance such as a macromolecular compound;
and/or the medicament comprises an animal and/or plant extract;
preferably, the small molecule compound comprises a pharmaceutically acceptable salt, solvate of a compound, solvate of a pharmaceutically acceptable salt of a compound, or crystalline form of a compound;
preferably, the medicament comprises an injectable formulation and/or an oral formulation.
4. Application of radix Pseudostellariae cyclic peptide B in preparing medicine for preventing and/or treating cardiomyopathy is provided.
5. The use according to claim 4, wherein the cardiomyopathy is diabetic cardiomyopathy.
6. The use according to claim 4, wherein the medicament is a medicament that binds mitochondrial antiviral signaling proteins;
preferably, the drug is a drug that activates mitochondrial antiviral signaling proteins;
preferably, the medicament is for preventing and/or treating cardiomyopathy by one or more of the following mechanisms:
(1) Inhibiting collagen formation in myocardial tissue;
(2) Maintaining mitochondrial homeostasis of cardiomyocytes;
(3) Maintaining mitochondrial autophagy homeostasis of cardiomyocytes;
(4) Inhibit apoptosis of cardiomyocytes.
7. The use according to any one of claims 4-6, wherein the medicament comprises a small molecule substance, e.g. a small molecule chemical such as a small molecule compound, or a small molecule biological such as a small molecule active peptide-conjugate;
and/or the drug comprises a macromolecular substance, for example a macromolecular biological substance such as an antibody-conjugate, or a macromolecular chemical substance such as a macromolecular compound;
and/or the medicament comprises an animal and/or plant extract;
preferably, the small molecule compound comprises a pharmaceutically acceptable salt, solvate of a compound, solvate of a pharmaceutically acceptable salt of a compound, or crystalline form of a compound;
preferably, the medicament comprises an injectable formulation and/or an oral formulation.
8. Use of a pharmaceutical formulation comprising pseudostellaria root cyclic peptide B, or a pharmaceutically acceptable salt thereof, a solvate of a compound, a solvate of a pharmaceutically acceptable salt of a compound, or a crystalline form of a compound, and at least one pharmaceutically acceptable carrier or excipient, for the preparation of a medicament for improving cardiac function;
preferably, the pharmaceutically acceptable carrier or excipient is selected from at least one of solvents, diluents, disintegrants, precipitation inhibitors, surfactants, glidants, binders, lubricants, dispersants, suspending agents, isotonic agents, thickening agents, emulsifiers, preservatives, stabilizers, hydration agents, emulsification accelerators, buffers, absorbents, colorants, fragrances, sweeteners, ion exchangers, mold release agents, coating agents, flavoring agents, and antioxidants;
preferably, the pseudostellaria root cyclic peptide B is provided in the form of a monomer or in the form of an animal and/or plant extract containing the same;
preferably, the formulation is an injectable formulation and/or an oral formulation.
9. Use of a pharmaceutical formulation for the preparation of a medicament for the prevention and/or treatment of cardiomyopathy, characterized in that the formulation comprises pseudostellaria root cyclic peptide B, or a pharmaceutically acceptable salt thereof, a solvate of a compound, a solvate of a pharmaceutically acceptable salt of a compound, or a crystalline form of a compound, and at least one pharmaceutically acceptable carrier or excipient;
preferably, the pharmaceutically acceptable carrier or excipient is selected from at least one of solvents, diluents, disintegrants, precipitation inhibitors, surfactants, glidants, binders, lubricants, dispersants, suspending agents, isotonic agents, thickening agents, emulsifiers, preservatives, stabilizers, hydration agents, emulsification accelerators, buffers, absorbents, colorants, fragrances, sweeteners, ion exchangers, mold release agents, coating agents, flavoring agents, and antioxidants;
preferably, the pseudostellaria root cyclic peptide B is provided in the form of a monomer or in the form of an animal and/or plant extract containing the same;
preferably, the formulation is an injectable formulation and/or an oral formulation.
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| CN107714914A (en) * | 2017-09-28 | 2018-02-23 | 李振升 | Treat Chinese medicine preparation of diabetic cardiomyopathy and preparation method thereof |
| CN110237232A (en) * | 2019-07-30 | 2019-09-17 | 广东海洋大学 | Radix pseudostellariae cyclic peptides B is improving the application in memory and/or senile dementia prevention and cure |
| CN112603978A (en) * | 2021-01-05 | 2021-04-06 | 中国中医科学院广安门医院 | Traditional Chinese medicine composition for treating type 2 diabetes combined with coronary heart disease and preparation method thereof |
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2023
- 2023-06-19 CN CN202310725355.6A patent/CN116617360A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107714914A (en) * | 2017-09-28 | 2018-02-23 | 李振升 | Treat Chinese medicine preparation of diabetic cardiomyopathy and preparation method thereof |
| CN110237232A (en) * | 2019-07-30 | 2019-09-17 | 广东海洋大学 | Radix pseudostellariae cyclic peptides B is improving the application in memory and/or senile dementia prevention and cure |
| CN112603978A (en) * | 2021-01-05 | 2021-04-06 | 中国中医科学院广安门医院 | Traditional Chinese medicine composition for treating type 2 diabetes combined with coronary heart disease and preparation method thereof |
Non-Patent Citations (2)
| Title |
|---|
| JING TIAN ET AL: "Shengmai San Alleviates Diabetic Cardiomyopathy Through Improvement of Mitochondrial Lipid Metabolic Disorder", 《CELLULAR PHYSIOLOGY AND BIOCHEMISTRY》, 31 December 2018 (2018-12-31) * |
| 朱可扬: "NOD1/RIP2介导的线粒体稳态在太子参环肽B抗糖尿病心肌病中的作用机制研究", 《MEDSCI梅斯》, 31 January 2022 (2022-01-31) * |
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