Traditional Chinese medicine composition and application thereof

Abstract

The invention belongs to the technical field of traditional Chinese medicines, and particularly relates to a traditional Chinese medicine composition and application thereof. The traditional Chinese medicine composition is prepared from the following raw material medicines in parts by weight: 12-15 parts of codonopsis pilosula, 12-15 parts of herba epimedii, 12-15 parts of golden cypress, 12-15 parts of angelica sinensis, 10-12 parts of ligusticum wallichii, 12-15 parts of radix paeoniae alba, 12-15 parts of rhizoma atractylodis and 28-33 parts of salvia miltiorrhiza. The traditional Chinese medicine composition can be used for preparing a medicine for treating cerebral arterial thrombosis-coronary heart disease.

Description

Technical Field

[0001] The present invention belongs to the technical field of traditional Chinese medicine, and particularly relates to a traditional Chinese medicine composition and its application. Background Art

[0002] Comorbidity refers to the state in which two or more chronic non-infectious diseases coexist simultaneously. A study involving more than 2 million people shows that the incidence of comorbidity is relatively high in Chinese middle-aged and elderly populations, reaching 51.6% and 81.3% respectively. Ischemic stroke (IS) complicated with coronary heart disease is a common type of brain-heart comorbidity in clinical practice. A real-world study involving 40,310 IS patients shows that 19.91% of IS patients also suffer from coronary heart disease, which is the most common comorbidity except for risk factor diseases such as hypertension and diabetes. As China gradually enters the aging population, stroke has become the leading cause of disability and death in China. Coronary heart disease is also the main cause of global disease death and disability. The mortality rate of coronary heart disease patients in China shows an upward trend, and the number of coronary heart disease patients will continue to grow rapidly in the next decade. Therefore, both IS and coronary heart disease have high incidence and high mortality rates. They have common risk factors and causes of disease, and can induce and exacerbate each other.

[0003] In Western medicine, the treatment of IS mainly focuses on various measures to improve cerebral blood circulation (such as thrombolysis, antiplatelet, anticoagulation, defibrination, volume expansion, etc.) and neuroprotection. Antiplatelet and anticoagulation treatments are mainly limited to secondary prevention and the prevention and treatment of deep vein thrombosis. The success rate of rescue with recombinant tissue plasminogen activator (rt-PA) intravenous thrombolysis and mechanical thrombectomy is closely related to the treatment time window. The treatment of coronary heart disease mainly focuses on drug treatment (nitrate drugs, β-blockers, calcium channel blockers, other anti-myocardial ischemia drugs, etc.) and other therapies to improve myocardial ischemia and reduce cardiac load. However, both medical drug treatment and revascularization treatment have side effects and certain limitations. The comorbidity of the two will cause an increase in the risk of multiple drug use, drug toxicity accumulation, etc., with greater risks and an increased economic burden.

[0004] In traditional Chinese medicine, IS belongs to the category of "stroke". Currently, most doctors have a relatively consistent understanding of its etiology and pathogenesis, mainly starting from deficiency, phlegm, stasis, wind, and blood. Coronary heart disease belongs to the category of "chest impediment", which can be divided into two types: excess syndrome (phlegm turbidity, qi stagnation, cold congelation, blood stasis) and deficiency syndrome (yin deficiency, qi deficiency, yang collapse, yang deficiency). Therefore, both are syndromes of deficiency and excess caused by deficiency of healthy qi, stasis of blood, and phlegm turbidity, with the disease locations in the brain and heart. Under the guidance of the theory of treating different diseases with the same method, they can be treated. Therefore, seeking an effective, safe, and economical traditional Chinese medicine treatment plan for brain-heart comorbidity is of great significance for the current clinical problems that urgently need to be solved.

[0005] In view of this, the present invention is specifically proposed. Summary of the Invention

[0006] The first object of the present invention is to provide a traditional Chinese medicine composition in view of the deficiencies in the prior art.

[0007] The second object of the present invention is the use of the traditional Chinese medicine composition.

[0008] To achieve the above first object, the technical solution adopted by the present invention is as follows:

[0009] A traditional Chinese medicine composition, the traditional Chinese medicine composition comprising active ingredients, the active ingredients being made from the following raw materials in parts by weight: 12-15 parts of Codonopsis pilosula, 12-15 parts of Epimedium brevicornu, 12-15 parts of Phellodendron amurense, 12-15 parts of Angelica sinensis, 10-12 parts of Ligusticum chuanxiong, 12-15 parts of Paeonia lactiflora, 12-15 parts of Atractylodes lancea, 28-33 parts of Salvia miltiorrhiza.

[0010] Further, the active ingredients are made from the following raw materials in parts by weight: 15 parts of Codonopsis pilosula, 12-15 parts of Epimedium brevicornu, 12-15 parts of Phellodendron amurense, 12-15 parts of Angelica sinensis, 10-12 parts of Ligusticum chuanxiong, 12-15 parts of Paeonia lactiflora, 15 parts of Atractylodes lancea, 30 parts of Salvia miltiorrhiza.

[0011] Even further, the active ingredients are made from the following raw materials in parts by weight: 15 parts of Codonopsis pilosula, 15 parts of Epimedium brevicornu, 15 parts of Phellodendron amurense, 15 parts of Angelica sinensis, 10 parts of Ligusticum chuanxiong, 15 parts of Paeonia lactiflora, 15 parts of Atractylodes lancea, 30 parts of Salvia miltiorrhiza.

[0012] Further, the traditional Chinese medicine composition further comprises excipients, the excipients including at least one of a filler, a binder, a disintegrant, a lubricant, an absorbent and a diluent.

[0013] Further, the dosage form of the traditional Chinese medicine composition is selected from any one of granules, ointments, pills, injections and oral liquids.

[0014] To achieve the above second object, the technical solution adopted by the present invention is as follows: Application of the traditional Chinese medicine composition in the preparation of a drug for preventing and / or treating cerebro-cardiac comorbidity.

[0015] Further, the cerebro-cardiac comorbidity is ischemic stroke-coronary heart disease comorbidity.

[0016] Further, the drug is used for the treatment of the recovery period of ischemic stroke-coronary heart disease comorbidity.

[0017] Further, the drug exerts its efficacy through at least one of the following brain-related effects:

[0018] (1) Reducing the Longa-Z neurological function score;

[0019] (2) Reducing the cerebral infarction area;

[0020] (3) Reduce the damage of nerve cells in the brain;

[0021] (4) Enhance the protein expression of AKT1, p-AKT1, PI3K and BCL2;

[0022] (5) Reduce the protein expression of TP53 and BAX;

[0023] And exert the medicinal effect through at least one of the following cardiac effects:

[0024] (1) Reduce the area of myocardial infarction;

[0025] (2) Reduce the damage of cardiomyocytes;

[0026] (3) Enhance the protein expression of AKT1, p-AKT1, PI3K and BCL2;

[0027] (4) Reduce the protein expression of TP53 and BAX.

[0028] The formula of the traditional Chinese medicine composition of the present invention is also called Shenling Baigui Formula (SLBGF). It is a clinical experience formula formed by the inventor through years of clinical diagnosis and treatment practice for the co-morbidity of the elderly heart and brain (ischemic stroke combined with coronary heart disease). This formula is mainly aimed at patients with deficiency in origin and excess in superficiality of the heart and brain co-morbidity. Based on the principle of treating both the principal and secondary aspects of the disease, the prescription includes 8 herbs, namely Codonopsis pilosula, Epimedium brevicornu, Salvia miltiorrhiza, Ligusticum chuanxiong, Phellodendron amurense, Angelica sinensis, Paeonia lactiflora, and Atractylodes lancea, which together exert the effects of supplementing qi and activating blood circulation, strengthening the spleen and tonifying the kidney, and resolving dampness and eliminating phlegm. In the formula of the present invention, the inventor attaches importance to qi and blood, believing that the occurrence and development of the co-morbidity of the heart and brain are closely related to qi and blood. The treatment should focus on regulating qi movement. Only when qi movement is smooth can qi and blood flow smoothly. Therefore, the monarch herbs are Codonopsis pilosula and Epimedium brevicornu, the minister herbs are Salvia miltiorrhiza, Ligusticum chuanxiong and Phellodendron amurense, and the assistant and guiding herbs are Angelica sinensis, Paeonia lactiflora and Atractylodes lancea. Among them, Codonopsis pilosula and Epimedium brevicornu have the effects of strengthening the spleen and tonifying the kidney, Codonopsis pilosula, Salvia miltiorrhiza, Ligusticum chuanxiong, Paeonia lactiflora and Angelica sinensis have the effects of supplementing qi and activating blood circulation, and Phellodendron amurense and Atractylodes lancea have the effects of resolving dampness and eliminating phlegm. The monarch herbs strengthen the spleen and tonify the kidney to promote the better supplementing of qi and activating blood circulation by the minister herbs, and at the same time promote the better exertion of the effects of resolving dampness and eliminating phlegm by the assistant and guiding herbs. By mainly tonifying deficiency and activating blood circulation, the treatment of the co-morbidity of the heart and brain is focused on the patients. During the clinical diagnosis and treatment process, the inventor found that especially in elderly patients, the effect of tonifying deficiency and activating blood circulation treatment with this formula during the recovery period of the co-morbidity of the heart and brain is very good.

[0029] The treatment idea of this formula is different from the traditional treatment idea of clearing heat and detoxifying, promoting blood circulation and removing stasis for cardiovascular and cerebrovascular diseases. This formula has better curative effects for the treatment of the co-morbidity of the heart and brain, especially during the recovery period of the co-morbidity of the heart and brain.

[0030] The applicant carried out a series of experimental studies with MCAO and ISO rat models as carriers and found that the present invention can exert the curative effect advantages through at least one of the following brain effects:

[0031] (1) Reduce the Longa-Z neurological function score;

[0032] (2) Reduce the cerebral infarction area;

[0033] (3) Reduce the damage of nerve cells;

[0034] (4) Enhance the protein expression of AKT1, p-AKT1, PI3K and BCL2;

[0035] (5) Reduce the protein expression of TP53 and BAX;

[0036] And exert the therapeutic advantage through at least one of the following cardiac effects:

[0037] (1) Reduce the area of myocardial infarction;

[0038] (2) Reduce the damage of myocardial cells;

[0039] (3) Enhance the protein expression of AKT1, p-AKT1, PI3K and BCL2;

[0040] (4) Reduce the protein expression of TP53 and BAX.

[0041] The above results all indicate that Shenling Baigui Formula can effectively treat the co-morbidity of heart and brain (ischemic stroke-coronary heart disease co-morbidity). Brief Description of the Drawings

[0042] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings required for the description of the specific embodiments or the prior art. Obviously, the following drawings are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0043] Figure 1 It is a graph of the survival curve of rats (A Log-rank; B Cox model);

[0044] Figure 2 It is a graph of the comparison of the body weights of rats at different time points between groups (A before intervention; B after intervention);

[0045] Figure 3 It is a graph of the comparison of the neurological function scores of rats between groups (A non-intervention; B 24 h after intervention; C 48 h after intervention; D 1 w after intervention; E 2 w after intervention);

[0046] Figure 4 It is a graph of the typical echocardiogram images of the hearts of each group of rats after intervention;

[0047] Figure 5Box plot (only significant annotations) of each parameter index of cardiac ultrasound in rats after intervention, where the vertical coordinates from left to right and top to bottom are cardiac output (unit: milliliters per minute), end-diastolic ventricular diameter (unit: millimeters), end-systolic ventricular diameter (unit: millimeters), fractional shortening (unit: percentage), ejection fraction (unit: percentage), stroke volume (unit: microliters), left ventricular anterior wall diastolic thickness (unit: millimeters), left ventricular anterior wall systolic thickness (unit: millimeters), left ventricular posterior wall diastolic thickness (unit: millimeters), left ventricular posterior wall systolic thickness (unit: millimeters), end-diastolic ventricular volume (unit: microliters), end-systolic ventricular volume (unit: microliters); heart rate (number of heartbeats per minute);

[0048] Figure 6 TTC staining results of the brains of 6 groups of rats;

[0049] Figure 7 Comparison of differences between groups in TTC staining of rat brains;

[0050] Figure 8 HE staining results of rat brains (×40) (A: blank group; B: sham operation group; C: model group; D: low-dose Shenling Baigui formula group; E: high-dose Shenling Baigui formula group; F: aspirin group);

[0051] Fig. 9 HE staining results of rat hearts (×40) (A: blank group; B: sham operation group; C: model group; D: low-dose Shenling Baigui formula group; E: high-dose Shenling Baigui formula group; F: aspirin group);

[0052] Fig.10 Immunohistochemical results of rat brains;

[0053] Fig.11 Immunohistochemical results of rat hearts;

[0054] Fig.12 Protein blot results of BAX, BCL2, TP53, PI3K, AKT1 and p-AKT1 detected by Western blot in rats (A brain tissue; B heart tissue);

[0055] Fig.13 Results of inter-group difference analysis of Western blot in rat brains (API3K; BAKT1; Cp-AKT1; DBAX; EBCL2; FBCL2 / BAX; GTP53); where each vertical coordinate is the normalized expression level (Expression);

[0056] Fig.14Results of inter-group difference analysis of Western blot in rat hearts (API3K; BAKT1; Cp-AKT1; DBAX; EBCL2; FBCL2 / BAX; GTP53) figure; where each vertical coordinate is the normalized expression level (Expression);

[0057] Note: (1) In the above figures, the three symbols *, #, and ^ represent the significance of differences in data comparison between corresponding groups, as follows: compared with the blank group or sham operation group, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001; compared with the model group, #P<0.05, ##P<0.01, P<0.001, #P<0.0001; for comparison between treatment groups, ^P<0.05, ^^P<0.01, ^^^P<0.001;

[0058] (2) In the above figures, in the legends containing group names or groupings, Con represents the blank group; Sham represents the sham operation group; Model represents the model group; Aspirin represents the aspirin group; LSLBG represents the low-dose Shenling Baigui Formula group; HSLBG represents the high-dose Shenling Baigui Formula group;

[0059] (3) In the above figures, the abscissas of the bar charts are all group names or groupings, from left to right are the blank group, sham operation group, model group, aspirin group, low-dose Shenling Baigui Formula group, and high-dose Shenling Baigui Formula group. Detailed implementation manners

[0060] To make the objectives, technical solutions, and advantages of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Those skilled in the art should understand that the described embodiments are only for helping to understand the present invention and should not be regarded as specific limitations on the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts fall within the scope of protection of the present invention. The process parameters not specified with specific conditions in the following embodiments usually follow conventional conditions.

[0061] In the ranges disclosed in the present invention, the endpoints and any values of the ranges are not limited to the exact ranges or values, and these ranges or values should be understood to include values close to these ranges or values. For numerical ranges, between the endpoint values of each range, between the endpoint values of each range and individual point values, and between individual point values, they can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be regarded as specifically disclosed in the present invention.

[0062] All Chinese medicinal materials used in the present invention comply with the description of the corresponding medicinal materials in the 2020 edition of the Chinese Pharmacopoeia, especially the medicinal parts and the content of the active ingredients. For example, the content of baohuoside I (C 27 H 30 O 10 ) shall not be less than 0.030%. The content of phellodendron alkali in Phellodendron alkali hydrochloride (C 20 H 23 NO4·HCl), shall not be less than 0.34%.

[0063] The Chinese medicine composition of the present invention can be prepared into the following dosage forms: granules, pastes, pills, injections, oral liquids, etc.

[0064] When preparing different dosage forms, corresponding auxiliary materials are added, such as fillers, binders, disintegrants, lubricants, absorbents and diluents, etc. The preparation of different dosage forms can be carried out by conventional methods in the art, which will not be described in detail here.

[0065] The raw materials of the granules used in the following examples are the corresponding medicinal pieces. The granules used in animal experiments are prepared by mixing the raw material pieces and using modern technology (such as water decoction, concentration, and drying) to form a granular preparation that can be taken directly.

[0066] The present invention is further described in detail below in conjunction with specific experiments and results.

[0067] Effect and mechanism of Shenlingbaigui prescription in treating ischemic stroke-coronary heart disease comorbidity

[0068] Animal Experimental Research

[0069] 1. Experimental process

[0070] 1 Preparation of Shenlingbai Guifang Granules and Positive Control Drugs

[0071] The Shenlingbaigui prescription used in this study consists of 15 g of Codonopsis pilosula, 15 g of Epimedium, 15 g of Phellodendron, 15 g of Angelica sinensis, 10 g of Ligusticum chuanxiong, 15 g of White Peony Root, 15 g of Atractylodes macrocephala, and 30 g of Salvia miltiorrhiza. One dose of granules is prepared from it and converted into equivalent granules. The corresponding masses of each substance are: 15 g of Codonopsis pilosula, 2.3 g of Epimedium, 3 g of Phellodendron chinense, 10 g of Angelica sinensis, 3.3 g of Ligusticum chuanxiong, 3.3 g of White Peony Root, 7.5 g of Atractylodes macrocephala, and 15 g of Salvia miltiorrhiza. The granules were provided by the granule pharmacy of the TCM outpatient department of the China Academy of Chinese Medical Sciences.

[0072] The Shenling Baigui Granules were brewed and stirred for dissolution with purified water at 90°C - 100°C according to the dispensing concentrations of low dose 385.71 mg / mL and high dose 771.43 mg / mL, that is, 1 dose of the low-dose granule solution was brewed with 154 mL of water, and 1 dose of the high-dose granule solution was brewed with 77 mL of water. Both the low-dose and high-dose granule solutions were at adult-use concentrations.

[0073] According to the conversion of the human equivalent dose (HED, Human Equivalent Dose), the mouse dose was obtained. The total mass of each dose of the equivalent Shenling Baigui formula granule was 59.4 grams (converted according to the ratio) (total weight of 8 kinds of medicinal material granules). The minimum adult dosage of the granule was 1 dose per day. According to a standard adult body weight of 70 kg, the conversion coefficient of rats was 6.2. According to the formula for body surface area conversion, the human dose could be converted to the equivalent dose of rats as 5.26 g / kg·d -1 , which was set as the low-dose group (concentration 350 mg / mL), and twice this dose was set as the high-dose group (10.52 g / kg·d -1 , concentration 700 mg / mL). The Shenling Baigui granule solution was directly infused into the stomach of rats with a gavage needle or syringe.

[0074] In this study, aspirin was used as a positive control drug. The aspirin tablets used were calculated according to the common rat dose of 50 mg / kg. The required aspirin powder was weighed (the drug was ground into powder), an appropriate amount of sterile normal saline was added, and it was fully dissolved with a manual stirrer to prepare a 10 mg / mL aspirin solution. If there were insoluble particles during the preparation process, the solution could be filtered with a 0.22 μm sterile filter to ensure the solution was clean and free of impurities. The dissolved aspirin solution was dispensed into clean solution bottles. Mark the concentration and date of the aspirin solution on the bottle. If it was not used immediately, the solution could be stored in a 4°C refrigerator. The aspirin solution was directly infused into the stomach of rats with a gavage needle or syringe.

[0075] 2 Experimental animals

[0076] 84 SPF-grade SD rats, 6 - 8 weeks old (purchased from Beijing Speyford Biotechnology Co., Ltd., with a body weight of 260 - 280 g), were raised in the Experimental Center of the China Academy of Chinese Medical Sciences. The environmental temperature was 22 - 25°C, and the humidity was 50% - 85%. They could freely access food and water. There was a 12-hour light and 12-hour dark cycle, good ventilation, and other interferences such as noise were avoided (the environmental conditions were provided by the laboratory animal house). All rats were marked with a marker pen, such as marking 1, 2, 3~84 on the tail of each rat. They ate and drank normally, and all animals were adaptively fed for 3 - 5 days. This animal experiment was carried out in accordance with the experimental code regulations.

[0077] 3 Grouping and administration of animal experiments

[0078] After adaptive feeding, the rats were divided into 6 groups according to the random number table method: blank group (Control group, abbreviated as Con), sham operation group (Sham group, abbreviated as Sham), (comorbid) model group (Model group, abbreviated as Model), low-dose Shenling Baigui formula group (LSLBGF group, abbreviated as LSLBG), high-dose Shenling Baigui formula group (HSLBGF group, abbreviated as HSLBG), and aspirin group (Aspirin group, abbreviated as Aspirin). There were 8 rats in each of the blank group and the sham operation group, and 17 rats in each of the remaining groups.

[0079] The blank group, the sham operation group, and the comorbid model group were given saline (12 mL / kg) by gavage. The low-dose Shenling Baigui formula group was given the human equivalent dose of Shenling Baigui formula granules (5.26 g / kg·d -1 ) by gavage. The high-dose Shenling Baigui formula group was given twice the dose of the low-dose group (10.52 g / kg·d -1 ) of Shenling Baigui formula granules. The positive western medicine blank group was given aspirin (50 mg / kg). Each group was given gavage once a day at 9:00 am. After the MCAO model was established, the drugs were administered for 2 weeks. At the same time, the coronary heart disease model was established for one week starting from the second week of drug administration. The rats were examined for the brain and heart from the initial drug administration to the end of two weeks of drug administration.

[0080] 4 Model preparation

[0081] 4.1 Preparation of MCAO model

[0082] Rats in the treatment groups (model group, low-dose Shenling Baigui formula group, high-dose Shenling Baigui formula group, and aspirin group) were selected and the middle cerebral artery occlusion (MCAO) was induced in rats using the suture method.

[0083] (1) Preparation of MCAO model: The MCAO rat model was prepared in the treatment groups using the modified suture method. The rats were fasted for 12 hours before surgery and were not deprived of water. The specific operation steps are as follows:

[0084] ① Anesthetize the rats by intraperitoneal injection of 1% sodium pentobarbital solution. Place the rats on the operating table, fix them with tape, take the supine position, cut off the hair on the neck, clean it up, fully expose the neck skin, and then disinfect it.

[0085] ②Make a longitudinal incision in the middle of the rat's neck, bluntly separate the muscles and fascia layer by layer, and then isolate the right common carotid artery (CCA), internal carotid artery (ICA), and external carotid artery (ECA). Artery clips are used for artery separation and clamping to reduce tissue damage.

[0086] ③Clamp the proximal end of the CCA and the distal end of the ICA, isolate and ligate the distal end of the ECA, make a "V" - shaped small incision at the front of the ligation point of the ECA, slowly insert the suture through the ECA into the ICA, open the artery clip at the distal end of the ICA, so that the suture enters the brain through the ICA. When encountering slight resistance (the key point where the suture reaches the middle cerebral artery), stop inserting the suture, and the insertion length is about 18 - 20 mm.

[0087] ④Fix the suture, ligate the ECA, suture the rat's neck skin layer by layer and disinfect, and expose about 1 cm of the suture outside the skin.

[0088] ⑤After 60 minutes of ischemia, open the suture, slowly pull out the exposed suture within 10 - 15 seconds to achieve ischemia - reperfusion, then cut off the excess part of the suture and suture the wound.

[0089] ⑦In the Sham group, only the CCA, ECA, and ICA are isolated, and no suture is inserted, and then sutured layer by layer. Moreover, in the Sham group, no ligation is performed on the blood vessels and no suture is inserted, and other operations are the same (for example, the treatment time of the Sham group should be consistent with that of the experimental group to exclude the interference of surgical time and anesthesia factors). After the operation, closely monitor the changes in the behaviors of the rats such as diet and movement, as well as possible problems such as abnormal body temperature and unconsciousness.

[0090] (2) Evaluation of paralysis degree after MCAO:

[0091] Refer to the Longa method to score at the time when the animals wake up spontaneously and at 24 hours (h), 48 h, 1 week (w), and 2 w after the operation (Longa - Z neurological function score):

[0092] 0 points: No signs of nerve injury; 1 point: Unable to fully straighten the contralateral forepaw; 2 points: The animal circles to the contralateral side (the frequency and time of circling can be recorded); 3 points: The animal topples to the contralateral side; 4 points: The animal loses consciousness and cannot walk spontaneously.

[0093] Animals with 1 point will recover quickly, and animals with 4 points will die in the short term. Therefore, the score of a successful animal model should be 2 - 3 points, representing moderate nerve injury.

[0094] 4.2 Preparation of a rat model of coronary heart disease

[0095] (1) Preparation of isoproterenol (ISO) injection

[0096] Isoproterenol hydrochloride (ISO) powder, mass: 5.00 g (produced by Shanghai Yuanye Bio-Technology Co., Ltd., product number: S31065g). Weigh it according to an injection concentration of 0.55 mg / mL and completely dissolve it in sterile normal saline (0.9% NaCl) at room temperature (20 - 25 °C). If there are insoluble particles, filter them using a 0.22 μm sterile filter to prepare an ISO solution.

[0097] (2) Preparation of coronary heart disease model

[0098] Select the rats in the treatment group that were successfully modeled by MCAO in Section 4.1 and had been administered drugs for one week. While administering drugs in the second week, intraperitoneally inject 2 mg / kg·d -1 ISO, and intraperitoneally inject 2 mL of ISO solution continuously for 7 days to induce a coronary heart disease (ISO) model of myocardial ischemia in rats.

[0099] (3) Evaluation of coronary heart disease rat model

[0100] Evaluating the ISO-induced coronary heart disease model by echocardiography is a very suitable method for non-invasive detection, which can monitor the changes in cardiac function and structure in real time.

[0101] ① Animal preparation: Rats should fast for 6 hours before echocardiography examination, but are allowed free access to water to reduce the impact of gastric distension on image clarity. Intraperitoneally inject a mild anesthetic (sodium pentobarbital 40 mg / kg) to reduce the activity of rats while maintaining their basic cardiac function unaffected by the anesthetic.

[0102] ② Equipment debugging: The echocardiograph vevo700 should be prepared and adjusted to the rat mode. Usually, a high-frequency probe (such as 10 - 14 MHz) is used to ensure the resolution of imaging the hearts of small animals. Ensure the correct position of the probe, usually placed on the left chest of the rat (near the ribs), and use enough ultrasonic coupling gel to ensure that there are no bubbles interfering with the conduction of ultrasonic waves. Gradually adjust the angle and position of the probe to obtain long-axis view and short-axis view to comprehensively observe the function of the left ventricle. According to the size and heart rate of the rat heart, the gain, depth, and frame rate can be further adjusted to optimize the resolution and clarity of the image. These parameter adjustments are crucial especially when evaluating cardiac systolic function (such as left ventricular ejection fraction).

[0103] ③ Measurement of key indicators:

[0104] Left ventricular ejection fraction (LVEF): This is a key indicator for evaluating left ventricular systolic function and usually decreases significantly when cardiac function declines. Calculation formula: LVEF = (left ventricular end-diastolic volume - left ventricular end-systolic volume) / left ventricular end-diastolic volume × 100%.

[0105] Left ventricular fractional shortening (LVFS): This indicator can also reflect the systolic ability of the left ventricle. Calculation formula: LVFS = (end-diastolic diameter - end-systolic diameter) / end-diastolic diameter × 100%.

[0106] Ventricular wall thickness: It is used to evaluate the degree of myocardial hypertrophy and myocardial fibrosis by ultrasound.

[0107] Cardiac output (CO): The overall pumping ability of the heart, which can be comprehensively evaluated in combination with the changes in the left ventricle.

[0108] ④ Successful model establishment: When the left ventricular infarction area reaches 30 - 40%, it is considered that the model construction is successful.

[0109] 5 Efficacy analysis

[0110] 5.1 Echocardiogram

[0111] As shown in the evaluation part of the coronary heart disease rat model in 4.2.

[0112] 5.2 Neurological function score

[0113] As shown in the evaluation of the degree of paralysis after MCAO in 4.1.

[0114] Scoring time:

[0115] 24 hours after surgery: After the rats wake up, a scoring evaluation is first performed.

[0116] 48 hours and 72 hours after surgery: In order to observe the recovery of the rats' neurological function, it is recommended to perform evaluations again 48 hours and 72 hours later respectively.

[0117] Observations at 1 week and 2 weeks after surgery: Observe the dynamic changes in the recovery or deterioration of neurological function.

[0118] 5.3 Sample collection

[0119] (1) Collection and processing of cardiac tissue

[0120] ① Anesthesia: Use 1 - 2% isoflurane inhalation anesthesia to ensure that the rats enter a state of deep anesthesia. Ensure that the rats are painless during sample collection.

[0121] ②Expose the heart: Fix the rat on the operating table. Use surgical scissors to cut open the skin and muscle along the midline of the chest, and bluntly separate layer by layer. Pay attention to separating the muscle tissue to avoid damaging internal organs such as the lungs and large blood vessels. Ensure that the entire thoracic cavity is fully exposed. Use scissors to cut open the sternum, expand the thoracic cavity, and expose the heart, ensuring that the surface of the heart is clean.

[0122] ③Cut off the blood vessels: Carefully cut the main blood vessels (such as the aorta and pulmonary artery) around the heart with surgical scissors. Avoid causing unnecessary damage to the heart tissue when cutting the blood vessels. Tweezers can be used to help separate and clamp the blood vessels to prevent excessive bleeding.

[0123] ④Drain the blood in the cavity: Place the heart in a beaker or petri dish containing phosphate-buffered saline (PBS), and gently squeeze the heart to make it contract on its own to drain the remaining blood in the cavity. This process needs to be carried out gently to maintain the structural integrity of the heart and avoid affecting subsequent tissue sectioning or protein extraction.

[0124] ⑤Absorb the liquid on the surface of the heart: Take out the heart and gently absorb the PBS solution and blood on the surface and in the cavity of the heart with sterile filter paper (manufacturer: Mabrey Membrane Technology Co., Ltd.). Ensure that the heart is dry for subsequent processing.

[0125] ⑥Tissue processing and preservation: According to experimental requirements, the heart can be processed and preserved in different ways:

[0126] Cryopreservation: For subsequent protein analysis such as WB (Western Blot), divide the heart into appropriate sizes, put it into a cryotube, and immediately store it in a -80°C refrigerator.

[0127] Fixation preservation: If histopathological analysis (such as HE staining) is required, place the heart in a 4% paraformaldehyde solution for fixation. Usually, the fixation time is 24 - 48 hours, and then transfer it to PBS solution for storage.

[0128] (2) Brain tissue collection and processing

[0129] ①Anesthetize and remove the brain: After anesthesia, quickly use surgical scissors to cut open the rat's scalp, cut and separate the skin and muscle tissues along the midline to expose the skull. Use a small saw or scissors to cut open the skull from the occipital bone and parietal bone, expand the incision until the brain is fully exposed, and pay attention to avoiding damaging the brain tissue. Use small tweezers to carefully remove the complete brain tissue, ensuring that all parts of the brain (cerebrum, cerebellum, and brainstem) remain intact.

[0130] ②Wash and drain: Place the removed brain tissue in PBS solution, gently shake it or moderately rinse it with tweezers to remove the blood and debris attached to the surface. Use sterile filter paper to absorb the moisture on the surface of the brain tissue to ensure that there is no residual liquid.

[0131] ③ Tissue processing and preservation: Brain tissue can be processed in different ways according to experimental requirements:

[0132] Cryopreservation: After the brain tissue or its different regions (such as cerebral cortex, hippocampus, etc.) are divided, placed in cryovials and immediately stored in a -80℃ refrigerator, which is suitable for protein extraction and WB analysis.

[0133] Fixation and preservation: If histopathological analysis or immunohistochemistry is required, the intact brain tissue or specific parts are fixed in 4% paraformaldehyde solution. The fixation time is usually 24 to 48 hours, and then transferred to PBS solution for preservation to avoid excessive fixation of the tissue, which may cause difficulties in subsequent experiments.

[0134] (3) Notes

[0135] Standardization of tissue cutting: To ensure comparability of subsequent experimental results, the cutting and partitioning of heart and brain tissues should be consistent and standardized using specific tools (such as slicing molds).

[0136] Aseptic technique: Maintain aseptic technique throughout the sampling process to avoid contamination, especially for tissues used for protein analysis.

[0137] Storage temperature control: Ensure that the -80℃ refrigerator and PBS solution are at the appropriate temperature and minimize exposure time during tissue processing to prevent protein degradation or tissue denaturation.

[0138] 5.4 Hematoxylin and Eosin (HE) staining of rat heart and brain pathological tissues

[0139] Hematoxylin: It is an alkaline dye that binds to DNA and RNA in the cell nucleus, staining the nucleus blue-purple. Since nucleic acids carry a negative charge, hematoxylin staining shows a clear outline of the cell nucleus. Eosin: It is an acidic dye that mainly binds to proteins in the cytoplasm and extracellular matrix, staining these areas pink or red. Therefore, non-nuclear structures such as the cytoplasm and connective tissue of the cell are usually stained red. HE staining is widely used to observe the morphological structure of tissues under a microscope. It can help researchers distinguish and identify different cell and tissue types for pathological analysis.

[0140] (1) Sampling and fixation:

[0141] Heart tissue: After the rat heart is removed, the surface and blood in the cavity are thoroughly cleaned with pre-cooled PBS solution. The surface moisture of the heart is dried with sterile filter paper. The heart is fixed in 4% paraformaldehyde solution for 24 hours.

[0142] Brain tissue: After removing the rat brain tissue, place the brain in cold PBS solution and gently rinse to remove blood. Blot the surface of the brain dry with filter paper and fix it in 4% paraformaldehyde solution for 24 hours.

[0143] (2) Dehydration, embedding and sectioning:

[0144] Gradient dehydration: The heart and brain tissues are gradually dehydrated in a gradient ethanol solution (70%, 80%, 90%, 100%) to remove the water in the tissues. Paraffin embedding: The dehydrated heart and brain tissues are embedded and fixed with paraffin to ensure the integrity of tissue sections. Paraffin sectioning: The embedded heart and brain tissues are respectively cut into 5-μm thick tissue sections on a paraffin microtome.

[0145] (3) Hydration and dewaxing:

[0146] Place the sections on glass slides and soak them in warm water at 40 °C to spread the sections. Treat the sections with xylene twice, 4 minutes each time, to remove the paraffin in the tissues. Then perform gradual hydration with a gradient ethanol solution (100%, 90%, 80%, 70%) to elute the xylene and fully hydrate the samples.

[0147] (4) Hematoxylin-eosin (HE) staining:

[0148] Hematoxylin staining: Place the hydrated sections in hematoxylin staining solution for 7 minutes and rinse with tap water for 10 minutes. Eosin staining: After soaking the sections in pure water for a few seconds, place them in eosin staining solution for 1.5 minutes and wash with PBS solution to remove the excess stain.

[0149] (5) Dehydration and clearing:

[0150] Gradient dehydration: Treat the sections with 70% ethanol, 80% ethanol, 90% ethanol and absolute ethanol for 10 seconds each to gradually dehydrate. Clearing treatment: Treat the dehydrated sections with xylene twice, 5 minutes each time, to make the sections transparent for subsequent observation.

[0151] (6) Mounting and observation

[0152] Mount the sections with neutral resin to ensure that the sections will not be oxidized by air after sealing. Place the sealed sections in a ventilated place to air dry naturally. Finally, observe the morphological structures of the heart and brain tissues under a microscope and take pictures for record.

[0153] 5.5 TTC staining of rat brain pathological tissue

[0154] TTC is reduced to red formazan under the action of mitochondrial dehydrogenase in living tissues. Therefore, normal tissues are stained red, while infarcted or necrotic tissues cannot reduce TTC due to loss of metabolic activity and appear white, enabling quantitative and qualitative analysis of the infarcted area.

[0155] (1) Specimen collection and freezing

[0156] Brain: After 24 hours of ischemia-reperfusion, SD rats were anesthetized with isoflurane and decapitated to obtain the brain. During the brain collection process, attention was paid to protecting the integrity of the brain. The brain was placed in a -30°C refrigerator and frozen for about 10 minutes, and then the brain was cut into 2-mm-thick coronal sections using a slicing mold.

[0157] (2) TTC staining

[0158] Brain staining: The brain sections were placed separately in physiological saline containing 2% TTC solution at 37°C and incubated in the dark for 30 minutes. The container was covered with tin foil to avoid the influence of light on the staining process. The container was gently shaken every 5 minutes to ensure sufficient staining of the sections. Normal tissues were stained red, while infarcted areas were white.

[0159] (3) Fixation and washing

[0160] ① Brain section washing: After staining, the sections were washed with PBS solution for about 3 minutes to remove excess staining agent.

[0161] ② Section fixation: The brain sections were fixed with 4% paraformaldehyde solution for 30 minutes to ensure the stability of the staining effect.

[0162] (4) Imaging and data processing

[0163] ① Imaging: The stained brain sections were neatly placed on a glass plate, and the staining results were photographed and recorded using a scanner or camera.

[0164] ② Data analysis: Using image processing software (such as ImageJ), the infarct volume of the brain was calculated. Calculation formula: Infarct volume (%) = infarct area volume / total tissue volume × 100%

[0165] 6 Immunohistochemical detection of PI3K, AKT1, p-AKT1, TP53, BAX, and BCL2 in the brain and heart 6.1 Experimental equipment

[0166] name factory model Dehydrator Wuhan Junjie Electronics Co., Ltd. JJ-12J Embedding machine Wuhan Junjie Electronics Co., Ltd. JB-P5 Pathology slicer Shanghai Leica Instruments Co., Ltd. RM2016 Frozen Table Wuhan Junjie Electronics Co., Ltd. JB-L5 Tissue spreading machine Zhejiang Jinhua Kedi Instrument Equipment Co., Ltd. KD-P oven Shanghai Huitai Instrument Manufacturing Co., Ltd. DHG-9140A Glass slides Jiangsu Shitai Experimental Equipment Co., Ltd. 1088105W Cover glass Jiangsu Shitai Experimental Equipment Co., Ltd. 10212432C Micro-wave oven Galanz Microwave Oven Appliance Co., Ltd. P70D20TL-P4 Decolorization shaker Beijing Liuyi Instrument Factory WD-9405A Vortex mixer Tianyue Electronics TYXH-II Pipette Big Dragon KE0003087 / KA0056573 Hematoxylin stain Wuhan Baiqiandu Biotechnology Co., Ltd. B1001 <![CDATA[H2O2]]> EZ2921B398 B12555 BSA (Bovine Serum Albumin) DAKO BIOFROXX DAB colorimetric kit Jiangsu Shitai Experimental Equipment Co., Ltd. 2005289 Neutral gum Wuhan Baiqiandu Biotechnology Co., Ltd. 10004160 Panoramic Scanner 3D

[0167] 6.2 Experimental reagents

[0168] Reagents factory Part Number Anhydrous ethanol Sinopharm Chemical Reagent Co., Ltd. Xylene Sinopharm Chemical Reagent Co., Ltd. EDTA (PH9.0) antigen retrieval solution Wuhan Baiqiandu Biological B2002 PBS buffer Solebao PN1020

[0169] 6.3 Experimental procedure

[0170] (1) Dewaxing of paraffin sections to water

[0171] Put the slices into xylene I for 15 min - xylene II for 15 min - anhydrous ethanol I for 5 min - anhydrous ethanol II for 5 min - 85% alcohol for 5 min - 75% alcohol for 5 min - and distilled water for washing.

[0172] (2) Antigen retrieval

[0173] Place the tissue sections in a repair box filled with EDTA antigen repair buffer (pH 9.0) in a microwave oven for antigen repair, high heat for 5 minutes, stop the fire for 5 minutes, and repeat 3 times. During this process, excessive evaporation of the buffer should be prevented and the slides should not be dried. After natural cooling, place the slides in PBS (pH 7.4) and shake on a decolorizing shaker to wash 3 times, each time for 5 minutes.

[0174] (3) Blocking

[0175] The sections were placed in 3% hydrogen peroxide solution and incubated at room temperature in the dark for 25 minutes to block endogenous peroxidase. The slides were then placed in PBS (pH 7.4) and washed on a decolorizing shaker for 3 times, 5 minutes each time.

[0176] (4) Draw a circle

[0177] After the slices are slightly dried, use a histochemical pen to draw circles around the tissue (to prevent the antibody from flowing away).

[0178] (5) Serum blocking

[0179] 3% BSA was added dropwise into the circle and incubated at room temperature for 30 min.

[0180] (6) Add primary antibody

[0181] Gently shake off the blocking solution, add the primary antibody prepared in a certain proportion of PBS to the slices, and place the slices flat in a humidified box and incubate at 4°C overnight. (Add a small amount of water to the humidified box to prevent the antibody from evaporating) See the table below:

[0182]

[0183]

[0184] (7) Add secondary antibody

[0185]

[0186] (8) DAB staining and counterstaining of cell nuclei

[0187] The slides were placed in PBS (pH 7.4) and washed on a decolorizing shaker for 3 times, 5 minutes each time. After the sections were slightly dried, DAB color developer was added to the circle and the color development was controlled by a microscope. After the color development was complete, the slides were rinsed with distilled water or tap water, counterstained with hematoxylin, differentiated with 1% hydrochloric acid alcohol (about 1 second), rinsed with tap water, blued with ammonia water, and rinsed with running water.

[0188] (9) Dehydration and sealing

[0189] Place the slices in 75% alcohol for 5 min-85% alcohol for 5 min-anhydrous ethanol I for 5 min-anhydrous ethanol II for 5 min-xylene I for 5 min to dehydrate and make them transparent. Take the slices out of xylene, dry them slightly, and seal them with neutral gum.

[0190] 7 Western Blot detection of brain and heart PI3K, AKT1, p-AKT1, TP53, BAX and BCL2

[0191] Take the rat's affected cerebral hemisphere, separate the cortex, or take the brain tissue and heart tissue and weigh them, add an appropriate amount of phosphate buffer (pH 7.4) to fully homogenize, centrifuge at 3600×g for 15 minutes, centrifuge radius 10cm, collect the supernatant, and store at -20℃ for testing. The detected proteins include: PI3K, AKT1, p-AKT1, TP53, BAX and BCL2.

[0192] 7.1 Experimental Instruments

[0193] Instrument Name brand model Electrophoresis Apparatus Bio-rad 1645070 Electroporator Bio-rad BE6085 pH Meter Metter-Toledo GmbH LP115 ELISA reader Biotek 800TS Fully automatic chemiluminescence image analysis system Tanon 5200

[0194] 7.2 Experimental consumables

[0195]

[0196]

[0197] 7.3 Experimental procedures

[0198] (1) Protein extraction

[0199] Rinse the tissue block 2-3 times with pre-cooled PBS buffer to remove blood stains, cut into small pieces and place in a homogenizer. Add 10 times the volume of tissue protein extraction reagent (add protease inhibitors within a few minutes before use) and thoroughly homogenize in an ice bath. Transfer the homogenate to a centrifuge tube and shake. Ice bath for 30 minutes, during which time use a pipette to repeatedly blow and beat to ensure that the homogenate is completely lysed. Centrifuge at 4℃12000rpm for 15 minutes and collect the supernatant, which is the total protein solution.

[0200] (2) Denaturation of total protein solution

[0201] Add 5×loading buffer to the total protein solution of each sample, vortex to mix well, heat at 98°C for 10 min. 30 s before heating, note that you need to open the lid and then close it to continue boiling. Pay attention to the viscosity of the protein after boiling. If it is relatively viscous, continue to boil the sample until it becomes watery.

[0202] ① For cell samples, centrifuge, vortex to mix well and then centrifuge again. Continue with the subsequent experiments. If not electrophoresing immediately, store at -80°C. After completely thawing before use, vortex to mix well, centrifuge and then load the samples.

[0203] ② For tissue samples, centrifuge, transfer the supernatant to a new EP tube, mix well and then centrifuge again. Continue with the subsequent experiments. If not electrophoresing immediately, store at -80°C. After completely thawing before use, vortex to mix well, centrifuge and then load the samples.

[0204] (3) Prepare the gel

[0205] Prepare 10% separating gel and 5% stacking gel according to the molecular weight of the protein. To prevent the gel from solidifying, pour the gel immediately after adding TEMED:

[0206] Separating gel formulation

[0207]

[0208] 5% stacking gel formulation

[0209]

[0210] (4) Pour the gel

[0211] Fix the clean glass plates, then add the pre-prepared separating gel and seal with water to remove air bubbles. After the gel solidifies, blot the water in the glass plates with filter paper, then add the stacking gel and insert the comb. Gently pull out the comb after the gel solidifies again;

[0212] (5) Electrophoresis

[0213] Start electrophoresis with the processed protein samples of each group. The voltage for the stacking gel is 80 V, and the voltage for the separating gel is 130 V. Terminate electrophoresis when the bromophenol blue migrates to the bottom of the gel.

[0214] (6) Transfer

[0215] Activate the PVDF membrane in methanol for 1 min. Place the black side of the clip in a glass petri dish filled with transfer buffer. From bottom to top, place a layer of sponge pad, three layers of filter paper, the separating gel, the PVDF membrane, three layers of filter paper, and finally cover with a layer of sponge pad. Remove air bubbles, clamp the clip, add pre-cooled transfer buffer, set the current to 250 mA, and transfer at ice bath for 1.0 h;

[0216] (7) Blocking

[0217] Put the PVDF membrane prepared in the previous step into an appropriate amount of 5% skim milk powder and incubate it on a shaker for 1 h for blocking;

[0218] (8) Primary antibody incubation and secondary antibody incubation

[0219] Dilute the primary antibody with the primary antibody diluent according to the ratio in the following table. Put the blocked PVDF membrane into a hybridization bag and incubate it overnight at 4°C. Wash it 5 times with TBST on a shaker, 5 min each time;

[0220] Dilute the secondary antibody with 5% skim milk powder according to the ratio in the following table. Incubate the membrane in the secondary antibody for 1 h. Wash it 5 times with TBST on a shaker, 5 min each time;

[0221]

[0222] (9) Luminescence detection

[0223] Drop the freshly prepared ECL mixed solution onto the protein side of the membrane for luminescence detection.

[0224] Adjust the exposure conditions according to different light intensities, develop and fix the film.

[0225] Result analysis: Scan and archive the film, and analyze the optical density value of the target band using the AlphaEaseFC software processing system.

[0226] II. Statistical analysis

[0227] Use R 4.3.2 software to analyze the data. The data are expressed as mean ± standard deviation (xˉ±s) or interquartile range. For measurement data that conform to normal distribution and have homogeneous variance, ANOVA is used for comparison among multiple groups, and Tukey or LSD test is used for pairwise comparison; for measurement data with repeated measurements, a general linear mixed-effects model is used for analysis. If the data are not normally distributed or have inhomogeneous variance, the Kruskal-Wallis H test is used for comparison among groups, and the Wilcoxon rank-sum test is used for pairwise comparison, with Bonferroni method correction. For survival analysis, the Kaplan-Meier method is used to plot the survival curve, and the Log-rank test is used for between-group differences; factors affecting survival prognosis are analyzed using the Cox regression model, and HR and 95% CI are calculated. All statistical analyses are completed using R 4.3.2, and P < 0.05 is considered statistically significant.

[0228] III. Results

[0229] 1 Comparison of the survival of rats

[0230] After MCAO and ISO model establishment, finally (two weeks after administration), all 8 rats in the blank group and 8 rats in the sham operation group survived. Among the model groups, 7 rats survived in the model group, 6 rats survived in the aspirin group, 7 rats survived in the low-dose Shenling Baigui formula group, and 11 rats survived in the high-dose Shenling Baigui formula group.

[0231] Survival analysis was performed on the survival status of rats in each group, and the Kaplan-Meier survival curve was drawn, as Figure 1 shown. Among them, the survival analysis based on the Log-rank test ( Figure 1 A) results showed that there were statistically significant differences in survival among the groups (Log-rank test, χ^2 = 17, df = 5, P = 0.0046). The blank group and the sham operation group had the best survival; the high-dose Shenling Baigui formula group was the second; the low-dose Shenling Baigui formula group was at the intermediate level; the survival rates of the model group and the aspirin group were relatively poor.

[0232] The survival analysis combining the Cox regression with Firth correction ( Figure 1 B) showed that there was no risk in the sham operation group and the blank group; the death risks of the model, aspirin, and low-dose Shenling Baigui formula groups were all significantly increased compared with the blank group, with HR = 14.87 (P = 0.0046), HR = 15.31 (P = 0.0037), and HR = 13.04 (P = 0.0081) in turn; however, the death risk of the high-dose Shenling Baigui formula group did not reach a statistically significant level compared with the blank group.

[0233] 2 Comparison of changes in rat body weight

[0234] In this study, statistical analysis was performed on the initial body weights of 6 groups of rats, and the results were as Figure 2 shown in A: The mean initial body weight of each group was between 266.0 g and 275.0 g; the p-values of the comparison results between the two groups were not significant after correction, indicating that the baseline body weights of each experimental group were balanced. In Figure 2 B, the body weight differences of rats in each group after model establishment and intervention were compared. The body weight levels between the blank group and the sham operation group were similar, without statistical differences. When the blank group and the sham operation group were respectively compared with the other 4 intervention groups, the differences were statistically significant (p.adj < 0.05), suggesting that the body weights of the blank group and the sham operation group were significantly higher than these four groups. In the pairwise comparisons between the treatment groups, all p-values were not statistically significant (p.adj > 0.05), suggesting that the body weight differences among these four groups were not significant.

[0235] 3.1 Results of neurological function scores

[0236] Longa-Z neurological function scores were performed at 0, 24 h, 48 h, 1 w, and 2 w after MCAO surgery without intervention, and the five scoring results as Figure 3 shown were obtained. Figure 3 As shown in A, the non-intervention scoring model group, aspirin group, high-dose Shenling Baigui formula group, and low-dose Shenling Baigui formula group all showed varying degrees of neurological impairment. However, there were no statistically significant differences among these four groups, indicating that the baseline of neurological function scores was comparable, and the modeling success rate was 82.35% (number of individuals with successful MCAO modeling / total number of individuals with MCAO modeling).

[0237] When measured at 24 h after intervention ( Figure 3 B), the Longa-Z scores of the model group, aspirin group, high-dose Shenling Baigui formula group, and low-dose Shenling Baigui formula group all decreased, suggesting that the neurological function of each treatment group had recovered to a certain extent after intervention. Within the treatment groups, there were significant differences between the model group and the high-dose Shenling Baigui formula group (P = 0.0017), and between the aspirin group and the high-dose Shenling Baigui formula group (P = 0.0114). There were no statistically significant differences among other treatment groups, indicating that the high-dose Shenling Baigui formula was effective relatively quickly.

[0238] In the measurement results at 48 h after intervention ( Figure 3 C), the scores of each treatment group further decreased compared with the previous time, indicating that the neurological function was continuously recovering. There were statistically significant differences between the model group and the aspirin group (P = 0.034), and between the model group and the low-dose Shenling Baigui formula group (P = 0.025). In particular, the difference between the model group and the high-dose Shenling Baigui formula group was the most obvious (P < 0.0001), indicating that the neurological function recovery of the model group was poor, while that of the high-dose Shenling Baigui formula group was good.

[0239] When measured at 1 w after intervention ( Figure 3 D), the scores of each treatment group continued to decrease, and the high-dose Shenling Baigui formula group continued to maintain the lowest level. Although the score of the model group improved further, it was still higher than that of the high-dose Shenling Baigui formula group and the low-dose Shenling Baigui formula group. There was a statistically significant difference between the high-dose Shenling Baigui formula group and the aspirin group, and the score of the high-dose Shenling Baigui formula group decreased significantly.

[0240] When measured at 2 w after intervention ( Figure 3 E), although the scores of the model group and the aspirin group decreased slightly compared with before; the scores of the high-dose Shenling Baigui formula group and the low-dose Shenling Baigui formula group were both less than 1 point, indicating that the neurological function had almost completely recovered. Statistical analysis showed that there were significant differences between the model group and the high- and low-dose Shenling Baigui formula groups (P < 0.01). However, there was no significant difference between the aspirin group and the model group.

[0241] 3.2 Results of cardiac ultrasound

[0242] In this study, cardiac systolic function, structural parameters, and ventricular wall thickness were evaluated by echocardiography system after intervention and ISO modeling. Figure 4 Shows typical echocardiogram images among different groups, Figure 5 showing the differences in echocardiographic indices among different intervention groups. Among them:

[0243] For heart rate, there were no statistical differences among the groups, indicating that the heart rates of the rats in each group were relatively stable during echocardiogram detection and the results were comparable.

[0244] Cardiac output and stroke volume (SV) were significantly lower in the model group than in the blank group (P<0.05), indicating reduced cardiac function in the model group; while there was a significant difference in SV between the blank group and the high-dose Shenling Baigui formula group (P<0.05), indicating reduced cardiac function in this group as well. There were no statistical differences in left ventricular end-diastolic and end-systolic volumes between the blank group and the sham operation group, while there were statistical differences between the blank group / sham operation group and other treatment groups. For left ventricular end-diastolic volume: P values for the blank group vs treatment groups (model group, aspirin group, low-dose Shenling Baigui formula group, and high-dose Shenling Baigui formula group) were 0.003, 0.003, 0.009, and 0.0003 respectively, and for the sham operation group vs treatment groups (model group, aspirin group, low-dose Shenling Baigui formula group, and high-dose Shenling Baigui formula group) were 0.003, 0.005, 0.009, and 0.001 respectively; for left ventricular end-systolic volume: P values for the blank group vs treatment groups (model group, aspirin group, low-dose Shenling Baigui formula group, and high-dose Shenling Baigui formula group) were 0.013, 0.0007, 0.0006, and 0.007 respectively, and for the sham operation group vs treatment groups (model group, aspirin group, low-dose Shenling Baigui formula group, and high-dose Shenling Baigui formula group) were 0.005, 0.003, 0.002, and 0.003 respectively, and the treatment groups were significantly lower. Combined with the reduction in cardiac output and stroke volume, it indicates that there may be a deterioration in myocardial compliance or a reduction in the overall ventricular volume.

[0245] The left ventricular end-diastolic diameter and end-systolic diameter were significantly smaller in the model group, aspirin group, low-dose Shenling Baigui formula group, and high-dose Shenling Baigui formula group than in the blank group / sham operation group. For the left ventricular end-diastolic diameter, the P values of the blank group vs. the treatment groups (model group, aspirin group, low-dose Shenling Baigui formula group, and high-dose Shenling Baigui formula group) were 0.003, 0.003, 0.009, and 0.0003 respectively, and the P values of the sham operation group vs. the treatment groups (model group, aspirin group, low-dose Shenling Baigui formula group, and high-dose Shenling Baigui formula group) were 0.003, 0.005, 0.009, and 0.001 respectively. For the left ventricular end-systolic diameter, the P values of the blank group vs. the treatment groups (model group, aspirin group, low-dose Shenling Baigui formula group, and high-dose Shenling Baigui formula group) were 0.013, 0.0007, 0.0006, and 0.007 respectively, and the P values of the sham operation group vs. the treatment groups (model group, aspirin group, low-dose Shenling Baigui formula group, and high-dose Shenling Baigui formula group) were 0.005, 0.003, 0.002, and 0.003 respectively, indicating a decrease in myocardial compliance and a compensatory increase in systolic ability. There were no statistical differences among the treatment groups. For the left ventricular ejection fraction (EF), the aspirin group (87.39%, IQR = 10.66) was higher than the model group (79.75%) and was significantly higher than the blank group (69.22%, P = 0.008) and the sham operation group (63.48%, P = 0.008). The EF of the aspirin group (87.39%) and the low-dose Shenling Baigui formula group (83.55%) were both higher than that of the model group, suggesting that it may relieve cardiac function injury by improving myocardial contractile efficiency, but it did not reach statistical significance. Although aspirin was superior to the Shenling Baigui formula group in improving systolic function (EF increased by 21.6%), the high-dose Shenling Baigui formula showed a potential dose-effect trend in reversing ventricular cavity narrowing (the left ventricular end-diastolic diameter increased to 6.435 mm).

[0246] The left ventricular fractional shortening rate was significantly higher in the aspirin group (58.466%) (P = 0.008) and the low-dose Shenling Baigui formula group (54.157) (P = 0.014) compared with the blank group. Compared with the sham operation group, the aspirin group (P = 0.003), the low-dose Shenling Baigui formula group (P = 0.004), and the high-dose Shenling Baigui formula group (P = 0.026) were all significantly increased. In addition, there were no statistically significant differences among the treatment groups (P > 0.05). Overall, aspirin and the low-dose Shenling Baigui formula were more significant in increasing the left ventricular fractional shortening rate, indicating that they have advantages in improving myocardial contractile function.

[0247] The diastolic thickness of the left ventricular posterior wall was significantly increased in the model group (2.251 mm) (vs the blank group 1.75 mm, P = 0.001), which was in line with the compensatory hypertrophy mechanism. The systolic thickness of the posterior wall was consistent with the diastolic thickness of the anterior wall. The diastolic thickness of the anterior wall was significantly increased in the model group (1.994 mm) (vs the blank group 1.71 mm; P = 0.02). The systolic thickness of the anterior wall in the model group (3.371 mm) was also significantly increased compared with that in the blank group (2.771 mm) (P = 0.013), indicating that the anterior wall also participated in the enhanced compensatory contraction. The diastolic thickness of the anterior wall was consistent with the systolic result.

[0248] 4 TTC staining results

[0249] The results of TTC staining of the rat brain were as Figure 6 , and it could be seen from the figure that no white cerebral infarction areas were found in the blank group and the sham operation group, while infarction areas were found in other groups. After 2 weeks of drug intervention, the infarction areas in the aspirin group and the high- and low-dose Shenling Baigui formula groups were all reduced to a certain extent, and the reduction in the high- and low-dose Shenling Baigui formula groups was more significant. One-way ANOVA was performed on the TTC results, and the results were as Figure 7 shown. There were extremely significant differences in the ratio of cerebral infarction area among different groups (P = 6.28×10-10), indicating that the experimental intervention had a significant effect on the range of brain tissue damage.

[0250] Figure 7 shown. The ratios of cerebral infarction area in the blank group and the sham operation group were significantly lower than those in the model group and the aspirin group, while the ratios of infarction area in the low-dose and high-dose Shenling Baigui formula groups were lower than that in the model group. Pairwise comparison between groups: The differences in the ratio of infarction area between the blank group and the treatment groups all reached a significant level (P < 0.01 for all), and the differences were particularly significant with the model group (P = 0.000593) and the aspirin group (P = 0.0000517). There was no significant difference in the ratio of infarction area between the sham operation group and the blank group (P = 0.465), but there were extremely significant differences compared with the model group, the aspirin group, and the high-dose Shenling Baigui formula group (P < 0.001), and there was an extremely significant difference compared with the low-dose Shenling Baigui formula group (P < 0.0001). There was a significant difference between the model group and the low-dose Shenling Baigui formula group (P < 0.05), and there was a significant difference between the model group and the high-dose Shenling Baigui formula group (P < 0.01), indicating that the intervention with high- and low-dose Shenling Baigui formula significantly reduced brain tissue damage. There were significant differences between the aspirin group and the low-dose Shenling Baigui formula group (P = 0.00490) and the high-dose Shenling Baigui formula group (P = 0.00782) (P < 0.01). There was no statistically significant difference between the low-dose and high-dose Shenling Baigui formula groups (P = 0.855), suggesting that the effects of the two doses of Shenling Baigui formula were similar in this index.

[0251] HE staining results

[0252] HE staining results of the brain( Figure 8 ): Figure 8 In the blank group of A and Figure 8 the sham operation group of B, the brain tissue showed no loosening, edema, softening foci, no swelling, degeneration or necrosis of neurons, normal perivascular and pericytic spaces, and no neuronophagia or satellitosis. Figure 8 In the model group of C, the brain tissue showed extensive structural loosening, disintegration, extensive neuronal degeneration and necrosis, accompanied by inflammatory cell infiltration; Figure 8 In the low-dose Shenling Baigui formula group of D and Figure 8 the aspirin group of F, the cell damage was relatively mild, and the number of intact cells increased significantly, while Figure 8 the high-dose Shenling Baigui formula group of E showed more improvement.

[0253] HE staining results of each group of the heart( Fig. 9 ): Fig. 9 In the blank group of A and Fig. 9 the sham operation group of B, the endocardium, myocardium and epicardium had clear structures, and no obvious abnormalities were found in the heart wall and heart cavity; the cross striations of cardiomyocytes were clear, alternating between light and dark, and the interstitial tissue was normal; no obvious inflammatory changes were observed; Fig. 9 In the model group of C, extensive ventricular damage occurred, the cross striations of myocardial fibers disappeared, cardiomyocytes swelled, were arranged disorderly, extensive necrosis and inflammatory cell infiltration occurred in the myocardial tissue, and a large amount of connective tissue hyperplasia was present between myocardial fibers; Fig. 9 In the low-dose Shenling Baigui formula group of D and Fig. 9 the aspirin group of F, the degree of cardiomyocyte damage was slightly less than that of the model group, and the number of intact cells increased, while Fig. 9 the high-dose Shenling Baigui formula group of E showed even less cardiomyocyte damage.

[0254] 6 Immunohistochemical results

[0255] By immunohistochemistry on the rat brain( Fig.10)Observation revealed that the brain tissue structure of the blank group and the sham operation group was intact, with orderly distribution of neurons, regular morphology, no obvious necrosis or inflammatory cell infiltration. The background staining of immunohistochemistry was clean. AKT1 was mainly located in the cytoplasm of neurons with weak staining intensity, and some astrocytes were light brown or negative; p-AKT1 was mainly located in the cytoplasm and perinuclear region of neurons, showing sporadic or weak positive; PI3K was mainly located in the cytoplasm of neurons, with a small number of glial cells showing weak positive; TP53 staining was sporadically weak positive, mainly seen in the nuclei or perinuclear regions of a few neurons; BAX and BCL2 also showed low levels of expression. In the infarction core area of the model group, neuronal degeneration, necrosis and vacuolar changes were visible, and glial cells and mononuclear macrophages were aggregated around blood vessels and the periphery of the infarct focus, and some cell nuclei were pyknosis or disappeared. Compared with the blank group and the sham operation group, the staining of AKT1, p-AKT1 and PI3K in the infarction core area and the penumbra area was significantly weakened in the treatment group; TP53 staining was enhanced, BAX expression was significantly up-regulated, and BCL2 expression was significantly decreased, suggesting an accelerated apoptosis process. Compared with the model group, the infarction area of the drug treatment group (aspirin group, low-dose Shenling Baigui formula group, high-dose Shenling Baigui formula group) was reduced, the degree of injury was relatively alleviated, and the neuronal morphology was relatively intact; the staining intensity of AKT1, p-AKT1 and PI3K was significantly enhanced, BAX staining was significantly weakened, the expression level of BCL2 was up-regulated, and at the same time the number of TP53 positive cells was also reduced. This trend was particularly significant in the high-dose Shenling Baigui formula group.

[0256] Observation was carried out on the rat heart by immunohistochemistry Fig.11 )and it was found that the myocardial fibers of the blank group and the sham operation group were arranged regularly, the number of interstitial cells was normal, AKT1, p-AKT1 and PI3K were mainly located in the cytoplasm and cell membrane of myocardial cells with weak expression intensity; TP53 was only sporadically weak positive; BAX and BCL2 were at the basal level. Compared with the blank group and the sham operation group, local necrosis and inflammatory cell infiltration occurred in the myocardium of the model group, and fibroblast hyperplasia was visible in the necrosis marginal area; the expression of AKT1, p-AKT1 and PI3K was enhanced in the damaged myocardial cells, TP53 and BAX were significantly increased, and BCL2 was relatively decreased, suggesting an accelerated apoptosis. Compared with the model group, the range of myocardial injury in the drug treatment group was reduced, the infiltration of inflammatory cells was decreased, and AKT1, p-AKT1 and PI3K still maintained relatively high or moderate expression levels; TP53 and BAX were significantly decreased, and BCL2 was up-regulated. Among them, the improvement in the high- and low-dose Shenling Baigui formula groups was more significant, indicating that it can effectively inhibit myocardial cell apoptosis and play a protective role.

[0257] 7 Results of Western Blot

[0258] The protein blots of BAX, BCL2, TP53, PI3K, AKT1, and p-AKT1 in the brain and heart tissues of mice were detected by Western blot as Fig.12 shown in A and 12B. The results of the inter-group difference analysis of Western blot in the rat brain are as Fig.13 shown, and the results of the inter-group difference analysis of Western blot in the rat heart are as Fig.14 shown.

[0259] Specifically, in the expression results of the PI3K-AKT pathway-related proteins in the brain tissue, as Fig.13 shown in A, 13B, and 13C, PI3K (0.186±0.078), AKT1 (0.231±0.092), and p-AKT1 (0.26±0.074) in the model group were significantly lower than those in the blank group and the sham operation group, and there were significant differences in PI3K and p-AKT1; in each dose group of Shenling Baigui Recipe, the expression levels of PI3K (high dose: 0.809±0.271; low dose: 0.557±0.137), AKT1 (high dose: 0.704±0.339; low dose: 0.541±0.208), and p-AKT1 (high dose: 0.804±0.076; low dose: 0.639±0.108) were significantly up-regulated compared with the model group, and there were significant differences in PI3K and p-AKT1, and the effect of the high dose group was more obvious. The expression levels of these three proteins in the aspirin group were higher than those in the model group, but there was no significant difference.

[0260] As Fig.13 shown in E, the expression of the anti-apoptotic protein BCL2 in the brain tissue of the model group (0.356±0.121) was significantly lower than that in the blank group (1±0.059) and the sham operation group (1.055±0.14); after treatment with Shenling Baigui Recipe, the expression level of BCL2 protein increased significantly, and the high and low dose groups (1.33±0.057, 0.767±0.121) were significantly higher than the model group, suggesting that Shenling Baigui Recipe may exert an anti-apoptotic effect by increasing the expression of BCL2. And Fig.13 in D, the expression of the pro-apoptotic protein BAX showed the opposite trend, with a significant increase in the model group (3.635±0.903), and a significant decrease in the high dose of Shenling Baigui Recipe (1.265±0.422) compared with the model group (P<0.05).

[0261] As Fig.13 shown in F, BCL2 / BAX in the aspirin group and the high and low dose groups of Shenling Baigui Recipe were significantly higher than the model group (P<0.05), and the aspirin group, low dose group of Shenling Baigui Recipe, and high dose group of Shenling Baigui Recipe showed an increasing trend. At the same time, as Fig.13As shown in Figure G, the expression of apoptosis-related protein TP53 in the model group (4.221±1.261) was significantly higher than that in the blank group and the sham operation group (P<0.05). After treatment with aspirin and Shenling Baigui formula, the level of TP53 decreased, but there was no statistical difference compared with the model group.

[0262] In the WB results of proteins related to the PI3K / AKT pathway in heart tissues ( Fig.14 ), the protein expressions of PI3K (0.206±0.092), AKT1 (0.218±0.072), and p-AKT1 (0.316±0.078) in the model group were significantly lower than those in the blank group (P<0.05), indicating that the PI3K / AKT pathway was inhibited. After treatment, the expressions of PI3K (0.74±0.245), AKT1 (0.823±0.233), and p-AKT1 (0.768±0.196) in the high-dose Shenling Baigui formula group were significantly higher than those in the model group, and the difference in p-AKT1 between the high-dose Shenling Baigui formula group and the model group was statistically significant (P<0.05); the expressions of PI3K (0.581±0.174), AKT1 (0.603±0.196), and p-AKT1 (0.611±0.114) in the low-dose Shenling Baigui formula group were significantly higher than those in the model group, and the differences in PI3K and p-AKT1 between the low-dose Shenling Baigui formula group and the model group were statistically significant (P<0.05). This shows that Shenling Baigui formula may participate in the mechanism of protecting the myocardium by activating the PI3K / AKT pathway, and the high-dose has a stronger effect, but there is no statistical difference between the high and low doses. Although the expression in the aspirin group was also higher than that in the model group, there was no significant difference compared with the model group.

[0263] Another example Fig.14As shown, the expressions of apoptosis-promoting proteins BAX (3.530±0.714) and TP53 (3.249±0.488) in the heart tissue of the model group were significantly higher than those in the blank group and the sham operation group, suggesting that the comorbidity model induced obvious cardiomyocyte apoptosis. However, the overall ANOVA test of the BAX protein did not reach statistical significance, while the inter-group difference of TP53 was statistically significant. The expression levels of BAX and TP53 proteins in each treatment group (treatment group) showed a decreasing trend. Among them, the decrease in the expression of BAX in the high-dose Shenling Baigui formula group (1.654±0.555) was more obvious, but there was no statistical difference among the groups; in terms of TP53 expression, the expressions in the high-dose Shenling Baigui formula (1.292±0.273) and low-dose Shenling Baigui formula (1.861±0.279) groups were significantly lower than those in the model group, indicating that Shenling Baigui formula may reduce myocardial apoptosis by inhibiting TP53 expression. At the same time, the expression of the anti-apoptotic protein BCL2 in the model group (0.383±0.099) was significantly decreased, with a significant difference from the blank group (P<0.05). The expressions of BCL2 in each dose of the Shenling Baigui formula group were significantly increased, especially in the high-dose group (1.471±0.281), with a statistical difference from the model group (P<0.01), suggesting that the treatment with Shenling Baigui formula may play a role in protecting cardiomyocytes by increasing BCL2 expression. The BCL2 / BAX in each treatment group was significantly higher than that in the model group, and showed an increasing trend in the aspirin group, low-dose Shenling Baigui formula group, and high-dose Shenling Baigui formula group.

[0264] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A traditional Chinese medicine composition, characterized in that: The traditional Chinese medicine composition comprises active ingredients, and the active ingredients are prepared from the following raw materials by weight: 12-15 parts of Codonopsis pilosula, 12-15 parts of Epimedium brevicornu, 12-15 parts of Phellodendron amurense, 12-15 parts of Angelica sinensis, 10-12 parts of Ligusticum chuanxiong, 12-15 parts of Paeonia lactiflora, 12-15 parts of Atractylodes lancea, 28-33 parts of Salvia miltiorrhiza.

2. The traditional Chinese medicine composition according to claim 1, wherein: The active ingredients are prepared from the following raw materials by weight: 15 parts of Codonopsis pilosula, 12-15 parts of Epimedium brevicornu, 12-15 parts of Phellodendron amurense, 12-15 parts of Angelica sinensis, 10-12 parts of Ligusticum chuanxiong, 12-15 parts of Paeonia lactiflora, 15 parts of Atractylodes lancea, 30 parts of Salvia miltiorrhiza.

3. The traditional Chinese medicine composition according to claim 2, characterized in that: The active ingredients are prepared from the following raw materials by weight: 15 parts of Codonopsis pilosula, 15 parts of Epimedium brevicornu, 15 parts of Phellodendron amurense, 15 parts of Angelica sinensis, 10 parts of Ligusticum chuanxiong, 15 parts of Paeonia lactiflora, 15 parts of Atractylodes lancea, 30 parts of Salvia miltiorrhiza.

4. The traditional Chinese medicine composition according to claim 1, wherein: The traditional Chinese medicine composition further comprises excipients, and the excipients include at least one of a filler, a binder, a disintegrant, a lubricant, an absorbent and a diluent.

5. The traditional Chinese medicine composition according to claim 1, wherein: The dosage form of the traditional Chinese medicine composition is selected from any one of granules, ointments, pills, injections and oral liquids.

6. Use of the traditional Chinese medicine composition according to any one of claims 1-5 in the preparation of a drug for preventing and / or treating cerebro-cardiac comorbidity.

7. The application according to claim 6, characterized in that: The cerebro-cardiac comorbidity is ischemic stroke-coronary heart disease comorbidity.

8. The application according to claim 6, wherein: The drug is used for the treatment of the recovery period of ischemic stroke-coronary heart disease comorbidity.

9. The application according to claim 6, wherein: The drug exerts its medicinal effects through at least one of the following brain-related effects: (1) Reducing the Longa-Z neurological function score; (2) Reducing the area of cerebral infarction; (3) Reducing the damage of nerve cells in the brain; (4) Enhancing the protein expression of AKT1, p-AKT1, PI3K and BCL2; (5) Reducing the protein expression of TP53 and BAX; And exerts its medicinal effects through at least one of the following heart-related effects: (1) Reducing the area of myocardial infarction; (2) Reducing the damage of myocardial cells; (3) Enhancing the protein expression of AKT1, p-AKT1, PI3K and BCL2; (4) Reducing the protein expression of TP53 and BAX.

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