CN111920871A

CN111920871A - Medicine for resisting myocardial ischemia reperfusion injury

Info

Publication number: CN111920871A
Application number: CN202010819648.7A
Authority: CN
Inventors: 蔡荣钦; 刘中秋; 陈婷; 程媛媛; 吴丽琼; 林琼英; 胡宏军; 陈思璇; 刘嘉铭; 杨莹
Original assignee: Guangdong Xinbao Pharmaceutical Technology Co ltd
Current assignee: Guangdong Xinbao Pharmaceutical Technology Co ltd
Priority date: 2020-08-14
Filing date: 2020-08-14
Publication date: 2020-11-13

Abstract

The invention provides a medicine for resisting myocardial ischemia reperfusion injury, which comprises Xinbao pills. The invention creatively discovers that the Xinbao pill can be used as a medicine for resisting myocardial ischemia reperfusion injury, and can also be used for preparing the medicine for resisting myocardial ischemia reperfusion injury, has strong protective effect on the anoxic reoxygenation h9c2 myocardial cells in vitro, and can inhibit the injury of the h9c2 cells caused by the anoxic reoxygenation; in vivo, the myocardial ischemia reperfusion injury induced myocardial infarction area and CK-MB content of a rat can be remarkably reduced by ligation of left anterior descending branch of coronary artery, and myocardial apoptosis is inhibited; meanwhile, the expression of apoptosis-related proteins of heart tissues can be inhibited, the proportion of anti-apoptosis proteins is increased, and the expression of autophagy-related proteins Beclin-1 and LC3II is inhibited. The invention provides a theoretical basis for researching the treatment strategy of myocardial ischemia-reperfusion injury and provides an insertion point for preparing a novel medicine for resisting myocardial ischemia-reperfusion injury.

Description

Medicine for resisting myocardial ischemia reperfusion injury

Technical Field

The invention belongs to the technical field of biological medicines, relates to a new application of a heart-nourishing pill medicine, and particularly relates to a medicine for resisting myocardial ischemia-reperfusion injury.

Background

Acute Myocardial Infarction (AMI) is a high risk, high mortality disease, and reperfusion is a common method in clinical settings. Reperfusion can effectively save surviving myocardial cells, reduce the area of an infarct area, maintain the contractile function of a left ventricle and block the occurrence of heart failure after blood supply is rapidly recovered, but in many cases, reperfusion often causes abnormal heart function, metabolism and structure, thereby aggravating cardiac tissue injury, namely Ischemia Reperfusion Injury (IRI). Therefore, prevention of myocardial Ischemia Reperfusion Injury (IRI) is a non-negligible clinical event for treating AMI. In order to reduce myocardial ischemia reperfusion injury, academia has found some treatment methods and drugs against the existing mechanism of IRI, such as Ischemic Preconditioning (IPC), remote ischemic conditioning, atorvastatin, etc. Although the surgical treatment methods have obvious effects, the operation is relatively complex; the above mentioned drugs are easy to use and show good pharmacodynamic action in animal experiments, but the results are not exact in clinical trial verification. Therefore, research and development of a medicament for reducing myocardial ischemia-reperfusion injury, which is simple to use and has a definite curative effect, are still necessary.

The Xinbao pill is a mature and commercially available traditional Chinese medicine compound preparation, which consists of nine traditional Chinese medicines of datura flower, hairy deerhorn, ginseng, monkshood, cinnamon, pseudo-ginseng, artificial musk, toad venom and borneol, is a black pill, each pill weighs 60mg, and the coating is removed to show tan, fragrant, sweet, slightly bitter and numb tongue feeling. The Xinbao pill has the functions of warming and invigorating heart and kidney, tonifying qi and supporting yang, and activating blood and promoting blood circulation, and can be used for treating chronic cardiac insufficiency caused by heart-kidney yang deficiency and heart vessel stasis, bradycardia caused by sinus node insufficiency, sick sinus syndrome and angina caused by ischemic heart disease and electrocardiogram ischemic change. Clinical research shows that the oral heart disease pill improves myocardial ischemia and cardiac function of patients with sick sinus syndrome and reduces ectopic premature beat; the Xinbao pill can prolong the left ventricular ejection time, shorten the PEP/LVET ratio, reduce the Ptfv1 negative value, improve the cardiac function, improve the heart failure symptom and adjust the tachycardia caused by cardiac insufficiency, and is an effective cardiotonic preparation. At present, no report that the Xinbao pill has the effect of resisting myocardial ischemia reperfusion injury in the prior art exists.

Disclosure of Invention

Aiming at the defects of the prior art, the invention aims to provide a new application of a heart disease treating pill.

In order to achieve the purpose, the invention adopts the following technical scheme:

in a first aspect, the invention provides a medicament for resisting myocardial ischemia-reperfusion injury, which comprises Xinbao pills.

In the invention, the medicament inhibits the reduction of the activity of h9c2 myocardial cells induced by hypoxia reoxygenation.

In the invention, the medicine inhibits myocardial ischemia reperfusion injury induced by ligation of left anterior descending branch of coronary artery.

In the present invention, the drug reduces the level of apoptosis-related protein caspase 3.

In the present invention, the drug increases the ratio of the levels of apoptosis-related protein Bcl2 to Bax.

In the invention, the medicament inhibits the expression of the autophagy-related protein Beclin-1.

In the present invention, the drug inhibits the expression of autophagy-related protein LC3 II.

The heart treasure pill related by the invention is a mature commercially available traditional Chinese medicine compound preparation, which is composed of nine traditional Chinese medicines of datura flower, pilose antler, ginseng, monkshood, cinnamon, pseudo-ginseng, artificial musk, toad venom and borneol, and is a black pill, wherein the weight of each pill is 60 mg. It can be prepared according to the preparation method recorded in heart treasure pill standard (WS3-B-3379-98) of eighteen books of Chinese medicinal prescription preparations of ministerial drug standards: respectively drying cornu Cervi Pantotrichum and part of Notoginseng radix, pulverizing into fine powder, and respectively pulverizing Bufonis venenum and Borneolum into fine powder; extracting cortex Cinnamomi by steam distillation, collecting volatile oil, decocting the residue for 2 times, filtering, mixing filtrates, concentrating to obtain soft extract, drying, and pulverizing into fine powder; extracting Ginseng radix with ethanol under reflux for three times, mixing extractive solutions, filtering, recovering ethanol, concentrating to obtain soft extract, drying, and pulverizing into fine powder; extracting flos Daturae Metelis with ethanol under reflux for three times, mixing extractive solutions, filtering, recovering ethanol, concentrating to obtain soft extract, drying, and pulverizing into fine powder; extracting part of Notoginseng radix with ethanol under reflux for three times, mixing extractive solutions, filtering, recovering ethanol, concentrating to obtain soft extract, drying, and pulverizing into fine powder; decocting radix Aconiti lateralis Preparata in water for 2 times, mixing decoctions, filtering, concentrating to obtain soft extract, drying, and pulverizing into fine powder. Mixing artificial Moschus with venenum Bufonis fine powder, Borneolum Syntheticum fine powder and part of Notoginseng radix fine powder, mixing with cortex Cinnamomi dry extract fine powder, Notoginseng radix dry extract fine powder and flos Daturae Metelis dry extract fine powder, mixing with Ginseng radix dry extract fine powder, radix Aconiti lateralis Preparata dry extract fine powder, cornu Cervi Pantotrichum fine powder and the rest Notoginseng radix fine powder, adding cortex Cinnamomi volatile oil and water, stirring to make soft material, making pill, drying, coating with appropriate amount of medicinal carbon and pulvis Talci, drying, polishing, and packaging.

The invention creatively discovers that the Xinbao pill can be used as a medicine for resisting myocardial ischemia reperfusion injury, has strong protective effect on the anoxic reoxygenation h9c2 myocardial cells in vitro, and can inhibit the injury of the h9c2 cells caused by the anoxic reoxygenation; in vivo, the myocardial ischemia reperfusion injury induced myocardial infarction area and CK-MB content of a rat can be remarkably reduced by ligation of left anterior descending branch of coronary artery, and myocardial apoptosis is inhibited; meanwhile, the expression of apoptosis-related proteins of heart tissues can be inhibited, the proportion of anti-apoptosis proteins is increased, and the expression of autophagy-related proteins Beclin-1 and LC3II is inhibited.

In a second aspect, the invention provides an application of Xinbao pills in preparing a medicament for resisting myocardial ischemia-reperfusion injury.

In a third aspect, the invention provides application of Xinbao pills in preparing a medicament for resisting hypoxia and reoxygenation injury of h9c2 cardiac muscle cells.

In a fourth aspect, the invention provides an application of Xinbao pills in preparing a medicament for resisting myocardial ischemia reperfusion injury induced by ligation of left anterior descending branch of coronary artery.

In a fifth aspect, the invention provides an application of Xinbao pill in preparing a medicament for inhibiting expression of apoptosis-related protein caspase 3.

In a sixth aspect, the invention provides application of Xinbao pills in preparation of a medicament for inhibiting expression of autophagy-related protein Beclin-1.

In a seventh aspect, the invention provides an application of Xinbao pill in preparing a medicament for inhibiting expression of autophagy-related protein LC3 II.

In the invention, the dosage form of the medicament comprises any one of suspension, granules, capsules, powder, tablets, emulsions, solutions, dripping pills, injections, suppositories, enemas, aerosols, patches or drops.

Preferably, the medicament further comprises pharmaceutically acceptable auxiliary materials.

Preferably, the auxiliary materials comprise any one or a combination of at least two of a carrier, a diluent, an excipient, a filler, a binder, a wetting agent, a disintegrating agent, an emulsifier, a cosolvent, a solubilizer, an osmotic pressure regulator, a surfactant, a coating material, a coloring agent, a pH regulator, an antioxidant, a bacteriostatic agent or a buffering agent. The combination of at least two of the above-mentioned components, such as the combination of diluent and excipient, the combination of binder and wetting agent, the combination of emulsifier and cosolvent, etc., can be selected in any combination manner, and will not be described in detail herein.

Compared with the prior art, the invention has the following beneficial effects:

the invention creatively discovers that the Xinbao pill can be used as a medicine for resisting myocardial ischemia reperfusion injury, and can also be used for preparing the medicine for resisting myocardial ischemia reperfusion injury, has strong protection effect on the h9c2 myocardial cells subjected to hypoxia reoxygenation in vitro, and can inhibit the h9c2 cell apoptosis caused by the hypoxia reoxygenation; in vivo, the myocardial ischemia reperfusion injury induced myocardial infarction area and CK-MB content of a rat can be remarkably reduced by ligation of left anterior descending branch of coronary artery, and myocardial apoptosis is inhibited; meanwhile, the expression of apoptosis-related proteins of heart tissues can be inhibited, the proportion of anti-apoptosis proteins is increased, and the expression of autophagy-related proteins Beclin-1 and LC3II is inhibited. The invention provides a theoretical basis for researching the treatment strategy of myocardial ischemia-reperfusion injury and provides an insertion point for preparing a novel medicine for resisting myocardial ischemia-reperfusion injury.

Drawings

FIG. 1 is a statistical chart of the results of the effect of Xinbao pills on the viability of h9c2 cardiomyocytes;

FIG. 2 is a statistical chart of the results of the effect of Xinbao pills on the viability of hypoxic reoxygenation h9c2 myocardial cells;

FIG. 3A is a graph of cross-sectional TTC staining results of three groups of rat hearts in example 3;

FIG. 3B is a statistical chart showing the results of the percentage of area of infarcted myocardium IR in the myocardium in the risk zone (IR/AAR) of two groups of rats in example 3;

FIG. 3C is a statistical chart showing the results of ELISA detection of the CK-MB content of three groups in example 3;

FIG. 3D is a graph of the results of HE staining of the heart of three groups of rats in example 3;

FIG. 4A is a graph showing the results of Western blot analysis of Caspase3, Bax, Bcl2, Tubulin, β -actin in example 4;

FIG. 4B is a graph showing the statistical effect of the groups on Caspase3 expression levels in example 4;

FIG. 4C is a graph of the statistical effect of groups on Bax/Bcl2 expression levels in example 4;

FIG. 5A is a graph showing the results of Western blot analysis of Beclin-1 and LC3II in example 5;

FIG. 5B is a graph showing the statistical effect of the groups in example 5 on the expression level of Beclin-1;

FIG. 5C is a graph of the statistical effect of groups on LC3II expression levels in example 5.

Detailed Description

The technical solution of the present invention is further explained by the following embodiments. It should be understood by those skilled in the art that the examples are only for the understanding of the present invention and should not be construed as the specific limitations of the present invention.

The procedures, conditions, reagents, test methods and the like for carrying out the present invention are those generally known in the art and are not specifically limited except for the contents specifically mentioned below. The test methods in each example, in which the specific conditions are not specified, are generally carried out under the conventional conditions or under the conditions recommended by the manufacturer.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs, but in the event of conflict, the present specification, including definitions, will control.

The heart-protecting pills used in the following examples were provided by Guangdong heart-protecting pharmaceutical science and technology Co., Ltd, and were manufactured in a batch number of 20170408, and were 60mg in weight per pill and 20 pills per bottle.

h9c2 rat cardiomyocytes were derived from the Shanghai academy of sciences cell bank.

Experimental animals adult male Sprague-Dawley (SD) rats (WM: 250-280 g) were purchased from southern university of medical laboratory animal center.

Example 1

Evaluation of Effect of Xinbao pills on h9c2 myocardial cell viability

The operation method comprises the following steps: taking h9c2 cardiomyocytes growing in logarithmic phase, inoculating the cardiomyocytes into a 96-well culture plate at 5000/well for overnight, adding complete DMEM medium containing Xinbao pills (0, 4, 8, 16, 32, 64, 128, 256, 512 and 1024 mu g/mL) with different concentrations after the cells grow well, and stopping culturing after 24 h. Then adding 100 mu L of 0.5mg/mL MTT solution, continuing to culture for 4h, discarding the culture medium, adding 150 mu L DMSO, shaking and mixing uniformly, placing on a microplate reader, and measuring the absorbance at 490 nm. The results are shown in FIG. 1: the Xinbao pill does not generate any toxicity to h9c2 myocardial cells under the concentration of 1024 mu g/mL.

Example 2

Evaluation of protective effect of Xinbao pill on hypoxia reoxygenation h9c2 cardiac muscle cells

The operation method comprises the following steps: taking h9c2 cardiomyocytes growing in logarithmic phase, inoculating the cardiomyocytes into a 96-well culture plate at 5000/well, standing overnight, after the cells grow well, replacing the cardiomyocytes with a sugar-free serum-free culture medium containing Xinbao pills (0, 10, 60, 240 and 720 mu g/mL) with different concentrations and a positive control drug diazoxide (100 mu M), placing the cardiocytes in an anaerobic device for culturing for 6h, replacing the completely normal DMEM culture medium, and stopping culturing after continuing to culture for 18 h. Then adding 100 mu L of 0.5mg/mL MTT solution, continuing to culture for 4h, discarding the culture medium, adding 150 mu L DMSO, shaking and mixing uniformly, placing on a microplate reader, and measuring the absorbance at 490 nm. The results are shown in FIG. 2: the Xinbao pill can obviously inhibit the activity reduction of h9c2 myocardial cells induced by hypoxia reoxygenation.

Example 3

The evaluation of the protective effect of the heart pill on myocardial ischemia reperfusion injury of rats induced by ligation of left anterior descending branch of coronary artery comprises the following contents:

(1) animal grouping: adult male Sprague-Dawley (SD) rats (WM: 250-280 g) were used for in vivo experiments. 4 rats were housed per cage and subjected to 12:12h light-dark cycles in an SPF animal house at 22 + -2 deg.C and humidity. Animals were allowed free access to standard rodent chow and distilled water. The 24 rats were randomly divided into 3 groups of 8 rats each: group 1 was the SHAM group (SHAM group), saline (i.p.); group 2 was MI/R group (myocardial ischemia reperfusion injury group), saline (i.p.); group 3 was MI/R + Heart treasure pill group (MI/R + XBW) (administered 180mg/kg, i.p.). The male SD rat is subjected to coronary artery left anterior descending ligation operation, and the Xinbao pill is dissolved in physiological saline and then is subjected to intragastric administration 1h before ligation. After 24h of reperfusion, cardiac ultrasound was detected and the rats were subjected to electrocardiographic testing, followed by euthanizing the rats and collecting samples.

(2) The construction method of the myocardial ischemia reperfusion model comprises the following steps: rats were anesthetized by intraperitoneal injection of 2% sodium pentobarbital (100 μ L/100g) and kept breathing with a ventilator. The rats were supine and the skin and pectoral muscles of their left breasts were cut open. Next, the intercostal space between the third and fourth ribs on the left side of the rat chest was cut open. The rat heart was exposed after opening the pericardium and the left anterior descending coronary artery (LAD) was ligated using 8-0 suture, placing a PE10 tube before ligation was complete. Successful ligation was confirmed by whitening of the anterior wall of the Left Ventricle (LV). At the same time, the thorax was closed quickly and closed with 4-0 sutures. After 30min of ischemia, the thorax was opened again, the PE10 tube was removed and the thorax was closed and the pectoralis muscles and skin were sutured. The operation is finished.

(3) Infarct size analysis method: left ventricular function was assessed by echocardiography 24h after MI/R. The rats were euthanized and venous blood was collected, the chest was opened, the left anterior descending coronary artery was ligated again, then 1% Evans blue dye was injected, the heart was removed and washed with PBS, frozen at-80 ℃ for 10min, and the cross section was cut into six pieces. The heart sections were placed in TTC and incubated in a dark room at 37 ℃ for 15min, then fixed with 4% paraformaldehyde overnight at 4 ℃. With the above treatment, blue is normal myocardium, pink is ischemic myocardium, and gray is necrotic myocardium, as shown in fig. 3A. The percentage area of infarcted myocardium IR in the myocardium at risk zone (IR/AAR) was calculated by Image J software and the results are shown in FIG. 3B: the Xinbao pill administration group (MI/R + XBW) can obviously reduce the area of myocardial infarction caused by myocardial ischemia reperfusion injury of rats.

(4) Detecting the content of CK-MB by ELISA: rat plasma was collected and assayed according to the protocol of rat CK-MB ELISA kit (manufactured by Nanjing grass Benyuan). The results are shown in FIG. 3C: the administration group of the heart pill (MI/R + XBW) can obviously reduce the CK-MB level.

(5) Hematoxylin-eosin staining: after euthanasia of the animals, hearts were collected and fixed in paraformaldehyde. After paraffin embedding, the sections (5 μm) were processed, deparaffinized, rehydrated, stained with hematoxylin-eosin (H & E), and photographed under an optical microscope. The results are shown in FIG. 3D: the heart nourishing pill administration group (MI/R + XBW) can reduce the pathological changes such as granulocyte infiltration, myocardial cell nucleus condensation and the like and reduce myocardial damage.

Example 4

Evaluation of Effect of Xinbao pill on apoptosis-related protein expression

The operation method comprises the following steps: approximately 250mg of rat left ventricle was placed in a centrifuge tube, and 500. mu.L of 1 XPPA buffer was added and ground with a grinder. Centrifuging at 12000rpm for 15min to obtain supernatant as extracted protein. After sample preparation, the samples were separated for different protein molecular weights by 10% or 15% SDS-PAGE at 100V constant voltage and placed in a Transfer Buffer (25mM Tris, 192mM glycine, 10% isopropanol) to electrotransfer the proteins onto polyvinylidene fluoride (PVDF) membranes at a constant current of 300 mA. After blocking the membranes for 1h with 5% BSA diluted in PBS-Tween 20 (0.1%) (PBST), the membranes were incubated overnight at 4 ℃ in specific antibodies (Caspase3, Bax, Bcl2, Tubulin, β -actin) (1/1000 in 2% BSA). Membranes were washed three times with PBS, incubated for 1h with secondary goat anti-rabbit or goat anti-mouse IgG antibodies (1/3000 in 2% BSA), and detected with chemiluminescence. The results are shown in FIGS. 4A, 4B and 4C: the Xinbao pill administration group (MI/R + XBW) can obviously reduce the level of apoptosis-related protein caspase3 and improve the Bcl2/Bax ratio, thereby resisting myocardial apoptosis and reducing reperfusion injury.

Example 5

Evaluation of the Effect of Xinbao pill on the expression of autophagy-related proteins Beclin-1 and LC3II

The operation method comprises the following steps: proteins were extracted from animal tissues and analyzed by western blot for expression of the autophagy-related proteins Beclin-1 and LC3II, in accordance with the method of example 4. The results are shown in FIGS. 5A, 5B and 5C: ischemia reperfusion can increase the expression of the autophagy-related protein Beclin-1, while the group administered with Xinbao pill can obviously reduce the expression of Beclin-1 and LC3 II.

The applicant states that the present invention is described by the above examples to describe a drug for resisting myocardial ischemia-reperfusion injury, but the present invention is not limited to the above examples, i.e. it does not mean that the present invention must be implemented by the above examples. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.

The preferred embodiments of the present invention have been described in detail, however, the present invention is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solution of the present invention within the technical idea of the present invention, and these simple modifications are within the protective scope of the present invention.

It should be noted that the various technical features described in the above embodiments can be combined in any suitable manner without contradiction, and the invention is not described in any way for the possible combinations in order to avoid unnecessary repetition.

Claims

1. The medicine for resisting myocardial ischemia reperfusion injury is characterized by comprising Xinbao pills.

2. The medicament of claim 1, wherein the medicament inhibits hypoxia-reoxygenation-induced decline in viability of h9c2 cardiomyocytes;

preferably, the medicament inhibits myocardial ischemia reperfusion injury induced by ligation of left anterior descending branch of coronary artery.

3. The medicament of claim 1 or 2, wherein the medicament reduces the level of apoptosis-related protein caspase 3;

preferably, the medicament increases the level ratio of apoptosis-related protein Bcl2 to Bax;

preferably, the drug inhibits the expression of the autophagy-related protein Beclin-1;

preferably, the drug inhibits the expression of autophagy-related protein LC3 II.

4. Application of XINBAO pill in preparing medicine for treating myocardial ischemia reperfusion injury is provided.

5. Application of Xinbao pill in preparing medicine for resisting h9c2 myocardial cell anoxia reoxygenation injury.

6. Application of Xinbao pill in preparing medicine for resisting myocardial ischemia reperfusion injury induced by ligation of left anterior descending branch of coronary artery.

7. Application of Xinbao pill in preparing medicine for inhibiting expression of apoptosis-related protein caspase 3.

8. Application of Xinbao pills in preparation of drugs for inhibiting expression of autophagy-related protein Beclin-1.

9. Application of Xinbao pill in preparing medicine for inhibiting expression of autophagy-related protein LC3 II.

10. The use of any one of claims 4 to 9, wherein the medicament is in a dosage form selected from the group consisting of a suspension, granules, a capsule, a powder, a tablet, an emulsion, a solution, a drop pill, an injection, a suppository, an enema, an aerosol, a patch or a drop;

preferably, the medicament further comprises pharmaceutically acceptable auxiliary materials;

preferably, the auxiliary materials comprise any one or a combination of at least two of a carrier, a diluent, an excipient, a filler, a binder, a wetting agent, a disintegrating agent, an emulsifier, a cosolvent, a solubilizer, an osmotic pressure regulator, a surfactant, a coating material, a coloring agent, a pH regulator, an antioxidant, a bacteriostatic agent or a buffering agent.