Application of tanshinol or pharmaceutically acceptable salt thereof in preparation of medicine for preventing, improving or treating coronary artery microvascular diseases

Abstract

The invention relates to the field of medical technology and food, in particular to application of tanshinol or pharmaceutically acceptable salt thereof in preparation of drugs for preventing, improving or treating coronary artery microvascular diseases. The invention provides application of tanshinol or pharmaceutically acceptable salt thereof in preparation of drugs for preventing, improving or treating coronary artery microvascular diseases. The traditional treatment limitation is broken through, and a novel high-efficiency low-toxicity medicine selection and treatment scheme is provided for prevention and treatment of CMVD.

Description

Technical Field

[0001] The present invention relates to the fields of medical technology and food, and in particular to use of danshensu or a pharmaceutically acceptable salt thereof in preparing a medicament for preventing, improving or treating coronary microvascular disease. Background Art

[0002] Cardiovascular disease (CVD) is a general term for cardiovascular diseases, including ischemic or hemorrhagic disorders of the heart and systemic tissues caused by conditions such as hyperlipidemia, blood viscosity, atherosclerosis, and hypertension. With high prevalence, disability, and mortality rates, CVD is one of the most lethal and life-threatening diseases worldwide.

[0003] Coronary microvascular disease (CMVD) refers to a clinical syndrome characterized by acute and chronic myocardial ischemia caused by structural and functional abnormalities of precoronary arterioles, arterioles, and capillaries, driven by both atherosclerotic and non-atherosclerotic pathogenic factors. Even in the early stages of coronary artery disease, before significant stenosis, CMVD can develop through mechanisms such as endothelial dysfunction, small vessel remodeling, inflammatory activation, and microthrombosis. This type of microvascular disease independently impacts myocardial blood supply and metabolic homeostasis, serving as a key prodromal condition for conditions such as angina pectoris, myocardial ischemia, and heart failure. It also underlies the pathogenesis of emerging heart failure phenotypes, including heart failure with preserved ejection fraction (HFpEF).

[0004] Traditional Chinese Medicine (TCM), with its thousands-year history of application in China, also plays a crucial role in the prevention and treatment of cardiovascular diseases. TCM emphasizes a holistic approach and individualized treatment. TCM believes that the development of cardiovascular disease is closely related to imbalances in Qi and blood, and in Yin and Yang. Herbs with the potential to promote blood circulation, dissipate stasis, relieve pain, and calm the liver and extinguish wind are commonly used in the prevention and treatment of cardiovascular diseases. These include Danshen (Salvia miltiorrhiza), Panax notoginseng (Panax notoginseng), Chuanxiong (Ligusticum chuanxiong), Curcuma aromatica (Curcuma), Millettia reticulata (Spatholobi), and Peach Kernel (Willow peach kernel), as well as Chinese patent medicines such as Compound Danshen Dripping Pills, Shensong Capsules, Naoxintong Capsules, and Musk Heart Protecting Pills.

[0005] Salvia miltiorrhiza (Danshen) is the dried root and rhizome of the Lamiaceae plant Salvia miltiorrhiza Bunge. It has the functions of removing blood stasis and relieving pain, promoting blood circulation and menstruation, and clearing the heart and relieving restlessness. It is commonly used to treat irregular menstruation, amenorrhea, dysmenorrhea, accumulation of lumps, chest and abdominal pain, restlessness and insomnia, angina pectoris, etc. Salvia miltiorrhiza has multiple pharmacological activities, including myocardial protection, vasodilation, anti-atherosclerosis, anti-thrombosis, regulation of tissue repair and regeneration, anti-inflammatory, and anti-tumor. Salvia miltiorrhiza has a clear therapeutic effect in the treatment of atherosclerosis. Its mechanism of action includes regulating endothelial cell function, inhibiting endothelial cell apoptosis, and protecting endothelial cells; regulating macrophage lipid metabolism, regulating macrophage phenotypic changes, and inhibiting foam cell formation; regulating inflammatory responses; and inhibiting the proliferation and migration of vascular smooth muscle cells. Danshensu is an important active ingredient in Danshen Root, exhibiting multiple pharmacological effects, including antioxidant, anti-inflammatory, and angiogenesis-promoting properties. Due to its structural characteristics, it primarily exists as a sodium salt, sodium danshensu. Upon entering the body, sodium danshensu hydrolyzes into sodium ions and danshensu, which then exerts its effects as danshensu. Although Danshen Root and its active ingredient, danshensu, have shown considerable potential in the prevention and treatment of cardiovascular diseases, there has been no research examining the application of danshensu and its salts in the prevention and treatment of coronary microvascular disease. This study focuses on the preventive and therapeutic effects of danshensu or its salts on coronary microvascular disease, aiming to provide novel, highly effective, and low-toxic drug options and treatment options for the prevention and treatment of CMVD. Summary of the Invention

[0006] The purpose of the present invention is to provide danshensu (C9H 10 O5) or its pharmaceutically acceptable salts in the prevention and treatment of coronary microvascular disease, for the first time expanding danshensu and its salt compounds to the field of CMVD prevention and treatment.

[0007] Danshensu and its sodium salt, Danshensu sodium, in the present invention are primarily derived from traditional Chinese medicines such as Danshen miltiorrhiza and can be obtained from methods such as extraction with ethanol, acetone, or pure water, or through necessary chemical synthesis. Danshensu sodium or other pharmaceutically acceptable salts of Danshensu in the present invention are stable chemical forms of Danshensu and can be decomposed into Danshensu upon entry into the body to exert their corresponding effects. Therefore, the activities and effects of Danshensu sodium discussed in the present invention can also be achieved in the form of Danshensu or other pharmaceutically acceptable salts of Danshensu.

[0008] The following is the technical solution of the present invention:

[0009] The present invention relates to the use of danshensu or a pharmaceutically acceptable salt thereof in preparing a medicament for preventing, improving or treating atherosclerosis-related coronary microcirculation disease, HFpEF-related coronary microvascular disease and diabetes-related coronary microvascular disease.

[0010] Furthermore, the diabetes is type 2 diabetes.

[0011] In the atherosclerosis-related coronary microvascular disease model, danshensu sodium can effectively reduce coronary blood flow reserve (CFR) and has an effective effect on improving microvascular perfusion function. In this model, danshensu sodium can also improve ventricular wall thickness and fibrosis area, effectively lower fasting blood sugar, improve glucose metabolism, increase serum HDL levels, and lower LDL, triglyceride (TG) and total cholesterol levels, showing the effect of lowering blood sugar and blood lipids; it can reduce the area of ​​atherosclerotic aorta and its branches, improve the degree of vascular stenosis, and inhibit the infiltration of foam cells and inflammatory cells in the plaque; danshensu sodium has the effect of promoting the formation of new blood vessels by promoting endothelial cell proliferation and migration.

[0012] In a model of HFpEF-related coronary microvascular disease, sodium danshensu can effectively reduce coronary flow reserve (CFR) and effectively improve microvascular perfusion function. In this model, sodium danshensu can also improve ventricular wall thickness and fibrosis, and restore arteriolar and capillary density, demonstrating its application value in promoting angiogenesis and alleviating microcirculatory remodeling. It can also increase serum HDL levels and reduce LDL, triglyceride (TG) and total cholesterol levels, demonstrating its blood sugar and lipid-lowering effects.

[0013] In the diabetes-related coronary microvascular disease model, sodium danshensu can effectively reduce coronary blood flow reserve (CFR) and effectively improve microvascular perfusion function; it also has the effect of improving cardiac vascular density and inhibiting the infiltration of cardiac macrophages.

[0014] The present invention also relates to the use of a pharmaceutical composition comprising danshensu or a pharmaceutically acceptable salt thereof in the preparation of a medicament for preventing or treating atherosclerosis-related coronary microcirculatory diseases, heart failure with preserved ejection fraction (HFpEF)-related coronary microvascular diseases, and diabetes-related coronary microvascular diseases, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable excipient and / or carrier, and the pharmaceutical composition comprises danshensu or a pharmaceutically acceptable salt thereof as an active ingredient. Furthermore, the diabetes is type 2 diabetes.

[0015] In the above uses, the excipients include any one or a combination of at least two of diluents, excipients, fillers, stabilizers, binders, wetting agents, disintegrants, emulsifiers, solubilizers, osmotic pressure regulators, surfactants, coating materials, pH regulators, antioxidants, antibacterial agents, colorants or buffers.

[0016] The drug involved in the present invention can be in the form of a preparation, or in the form of a food or health food, including tablets, granules, capsules, pills, oral liquids, injections, etc., and is not limited to the above-mentioned pharmaceutical dosage forms. It can be herbal tea, brewed beverages, solid food, etc., and is not limited to the above-mentioned forms.

[0017] The research results of the present invention show that danshensu or a pharmaceutically acceptable salt thereof has a good effect of preventing, improving or treating coronary microvascular disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Danshensu sodium reduces myocardial hypertrophy and fibrosis in mice with atherosclerosis-associated CMVD model. A: Comparison of body weight among mice in each group; B: Comparison of heart weight among mice in each group; C: Comparison of heart weight to body weight ratio among mice in each group; D: Representative images of heart HE staining (image scale: 1 mm); E: Representative images of heart Masson staining (image scale: 1 mm); F: Representative images of heart WGA fluorescence staining (image scale: 200 μm); G: Comparison of the degree of cardiac fibrosis among mice in each group; H: Comparison of cardiac cross-sectional area among mice in each group.

[0019] Figure 2 Danshensu sodium improves metabolic parameters in atherosclerosis-related CMVD model mice. A: Comparison of fasting blood glucose among mice; B: Comparison of serum insulin among mice; C: Comparison of serum HDL among mice; D: Comparison of serum LDL among mice; E: Comparison of serum TG among mice; F: Comparison of serum cholesterol among mice.

[0020] Figure 3 Danshensu sodium reduces atherosclerotic plaque formation in atherosclerosis-related CMVD model mice. A: Representative images of aorta stained with Oil Red (1 mm); B: Quantification of total aortic plaque area in each group of mice; C: Representative images of aorta stained with HE (150 μm); D: Comparison of atherosclerotic plaque area in each group of mice.

[0021] Figure 4 Danshensu sodium improves coronary blood flow reserve in atherosclerosis-related CMVD model mice: comparison of CFR among mice groups.

[0022] Figure 5 Danshensu sodium promotes angiogenesis in atherosclerosis-related CMVD model mice. A: Representative immunofluorescence staining images of CD31, α-SMA, and DAPI in the hearts of mice from each group (image scale: 2 mm); B: Comparison of cardiac capillaries in mice from each group; C: Comparison of cardiac arterioles in mice from each group.

[0023] Figure 6 Danshensu sodium improves basal metabolic parameters in mice with HFpEF-related CMVD. A: Comparison of body weight among mice in each group; B: Comparison of tail-cuff blood pressure among mice in each group; C: Comparison of fasting blood glucose among mice in each group; D: Comparison of serum insulin among mice in each group; E: Comparison of serum HDL among mice in each group; F: Comparison of serum LDL among mice in each group; G: Comparison of serum TG among mice in each group; H: Comparison of serum cholesterol among mice in each group.

[0024] Figure 7 Danshensu sodium alleviates myocardial fibrosis and decreased vascular density in mice with HFpEF-associated CMVD. A: Representative images of Masson staining of the hearts of mice in each group; B: Representative images of WGA staining of the hearts of mice in each group; C: Representative immunofluorescence images of α-SMA in the hearts of mice in each group; D: Representative immunofluorescence images of CD31 in the hearts of mice in each group; E: Comparison of the degree of cardiac fibrosis in the mice in each group; F: Comparison of the cross-sectional area of ​​myocardial cells in the mice in each group; G: Comparison of cardiac arterioles in the mice in each group; H: Comparison of cardiac capillaries in the mice in each group.

[0025] Figure 8 Danshensu sodium improves cardiac diastolic function in mice with HFpEF-related CMVD. A: Representative echocardiographic images of mice in each group; BC: Comparison of cardiac diastolic function indicators E / A and E / e' in each group.

[0026] Figure 9 Danshensu sodium restores coronary flow reserve in HFpEF-related CMVD model mice: representative echocardiographic images and quantitative comparison of CFR in each group of mice.

[0027] Figure 10 Changes in CFR in T2DM-related CMVD model mice after tanshinone sodium treatment.

[0028] Figure 11 Changes in cardiac vascular density and macrophage infiltration in T2DM-related CMVD model mice after tanshinone sodium treatment. DETAILED DESCRIPTION

[0029] The following examples illustrate specific implementation methods, further illustrating and supplementing the present invention. However, the scope of the present invention is not limited to the following examples. All technologies and applications based on the above-mentioned content of the present invention fall within the scope of the present invention.

[0030] Example 1 - Preparation of Danshensu Sodium Medicinal Tablets

[0031] Take 2g of sodium danshensu, 20g of dextrin, an appropriate amount of dry starch, an appropriate amount of carboxymethyl cellulose, an appropriate amount of talc, and an appropriate amount of sucrose. The sucrose and talc are used for sugar coating, mixed, granulated, and compressed into tablets containing 50mg of sodium danshensu per tablet. Other requirements must comply with the relevant provisions for tablets in the 2020 edition of the Pharmacopoeia of the People's Republic of China.

[0032] Example 2 - Preparation of Danshensu Sodium Medicinal Capsules

[0033] Mix 2g of danshensu sodium, 20g of corn starch, and an appropriate amount of cyclodextrin, and place into capsules. Each capsule contains 50-100mg of danshensu sodium. Other requirements must comply with the relevant provisions for capsules in the 2020 edition of the Pharmacopoeia of the People's Republic of China.

[0034] Example 3 - Preparation of Danshensu Sodium Sustained-Release Tablets

[0035] Mix 500g of sodium danshensu, 350g of carboxypropyl methylcellulose K100M, and 100g of microcrystalline cellulose. Prepare a 5% solution of 40g of povidone K30 with a 40% ethanol solution. Prepare a soft material, granulate with a 30-mesh sieve, dry at 50°C for 30 minutes, sieve through a 30-mesh sieve, add 10g of magnesium stearate, mix well, and press into tablets. Each tablet contains 50-500mg of sodium danshensu. Other requirements must comply with the relevant provisions for tablets in the 2020 edition of the Pharmacopoeia of the People's Republic of China.

[0036] Example 4 - Preparation of a physiological saline solution of sodium danshensu

[0037] Weigh danshensu sodium, add appropriate amount of normal saline, dissolve with ultrasonic aid and adjust to volume. Prepare immediately before use or store at 4℃ for a short period of time.

[0038] The physiological saline solution of sodium danshensu was used in the following animal experiments: an atherosclerosis-related coronary microvascular disease mouse model, a HFpEF-related coronary microvascular disease mouse model, and a T2DM-related coronary microvascular disease mouse model.

[0039] 1. Animal Experiment on the Effect of Danshensu Sodium on Alleviating Atherosclerosis-Related Coronary Microvascular Disease

[0040] 1. Medicines

[0041] Danshensu sodium: provided by Shanghai Qiguang Pharmaceutical Co., Ltd., pale white powder, content>98.0% (HPLC).

[0042] 2. Experimental Animals

[0043] The experimental animals were 8-week-old male Apolipoprotein E knockout mice (Apolipoprotein E knockout, APOE-KO), weighing approximately 20-22g, in good health, and the experiment began after one week of adaptive feeding. All animals were housed in a barrier system animal room with constant temperature and humidity (22±2°C, humidity 50-60%), with a circadian rhythm of 12 hours of light and dark cycle, and were allowed to drink water and eat freely. The control group mice were fed with standard laboratory ordinary chow, and the remaining mice were used to construct a coronary microvascular disease model (high-fat diet, HFD). All animal experimental operations followed the animal experimental operation procedures approved by the "Regulations on the Management of Laboratory Animals".

[0044] The experimental animals were randomly divided into 4 groups, with 8 mice in each group (n=8):

[0045] Control group (Control): standard diet, no high-fat feeding, gavage with normal saline;

[0046] Model group: model mice, gavage with normal saline;

[0047] Low-dose sodium danshensu group: model mice were gavaged with 150 mg / kg / day sodium danshensu;

[0048] High-dose sodium tanshinone group: Model mice were gavaged with 450 mg / kg / day of sodium tanshinone.

[0049] Drugs were administered orally starting from the 12th week and continued for 4 weeks until the 16th week of the experiment. During this period, drugs were administered at a fixed time of 9:00-10:00 am every day.

[0050] 3. Detection indicators and analysis methods

[0051] (1) Metabolic-related indicators

[0052] At the end of the experiment, fasting blood samples were collected from mice (after a 12-hour fast) and measured using an automated biochemical analyzer for metabolic markers such as fasting blood glucose, serum insulin, total cholesterol (TC), triglycerides (TG), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C). After excision, the heart was accurately weighed to calculate the heart weight / body weight ratio and assess cardiac hypertrophy.

[0053] (2) Assessment of the degree of atherosclerosis

[0054] Tissue from the aortic arch and thoracic aorta was obtained and stained with Oil Red O to observe lipid deposition and atherosclerotic plaque formation. Images were quantitatively analyzed using ImageJ software to determine the ratio of lipid plaque area to total vessel area, which was used as an indicator of atherosclerosis severity.

[0055] (3) Cardiac microcirculation function assessment

[0056] Coronary microcirculatory function was assessed using the CFR method. Ultrasound was used to measure the blood flow velocity ratio of the left anterior descending coronary artery at rest and after adenosine loading (140 μg / kg / min). H&E and Masson staining were also used to observe changes in myocardial tissue structure and the degree of fibrosis.

[0057] (4) Assessment of vascular density and inflammatory infiltration

[0058] Immunohistochemistry was used to assess myocardial angiogenesis and inflammatory responses. Antibodies against CD31 were used to label small vessel density, α-SMA to label arterioles, and VEGF to assess angiogenesis. Antibodies against CD45 and CD68 were used to identify the number of infiltrating leukocytes and macrophages, respectively, reflecting the intensity of the local inflammatory response. The staining results were photographed under a microscope and semi-quantitatively analyzed using Image-Pro Plus.

[0059] (5) Histopathological examination

[0060] Heart paraffin sections were stained with H&E to observe the overall tissue structural changes, and Masson staining was used to evaluate the degree of myocardial interstitial fiber deposition.

[0061] 4. Experimental Results

[0062] (1) Danshensu sodium improves cardiac structure and diastolic function in model mice

[0063] Danshensu sodium treatment significantly alleviated myocardial structural remodeling and functional abnormalities in model mice. After treatment, the mice's heart weight / body weight ratio decreased, and ventricular hypertrophy was alleviated. Histological analysis showed that the cross-sectional area of ​​myocardial cells decreased under HE staining, Masson staining indicated a decrease in the degree of interstitial fibrosis, and WGA staining revealed a more regular myocardial arrangement. The high-dose group was particularly effective in improving indicators such as ventricular wall thickness and fibrosis area. Figure 1 )

[0064] (2) Danshensu sodium improves metabolic disorders in model mice

[0065] Model mice often experience significant metabolic abnormalities, including elevated fasting blood glucose and impaired glucose tolerance. Low- and high-dose intervention with danshensu sodium can effectively lower fasting blood glucose and improve glucose metabolism. Insulin ELISA results combined with blood glucose level analysis suggest that insulin resistance has been alleviated. Figure 2 )

[0066] (3) Danshensu sodium improves the lipid profile of model mice

[0067] The model mice showed typical dyslipidemia, including elevated total cholesterol, triglycerides, and low-density lipoprotein (LDL-C), and decreased high-density lipoprotein (HDL-C). Danshensu sodium treatment significantly improved the lipid profile, with the high-dose group showing the best effect in lowering LDL-C, TG, and increasing HDL-C. Danshensu sodium has a clear advantage in lipid regulation. Figure 2 )

[0068] (4) Danshensu sodium inhibits the formation of atherosclerotic plaques

[0069] Oil red O and HE staining revealed significant lipid deposition and vascular wall thickening in the aorta and its branches in the model mice, suggesting significant atherosclerosis. Low- and high-dose intervention with tanshinone sodium significantly reduced plaque area, improved the degree of vascular stenosis, and inhibited the infiltration of foam cells and inflammatory cells within the plaques. Figure 3 )

[0070] (5) Danshensu sodium improves coronary microcirculatory function (CFR)

[0071] The CFR of the model mice decreased significantly (CFR < 2.0), indicating that the coronary artery lacked sufficient dilation capacity under oxygen demand. After intervention with sodium tanshinone, the CFR was significantly restored, suggesting that it can effectively improve microvascular perfusion function. Figure 4 )

[0072] (6) Danshensu sodium promotes angiogenesis

[0073] The density of cardiac capillaries and arterioles and the number of newly formed blood vessels were assessed by immunofluorescence staining of mouse heart tissue. Figure 5 A shows representative immunofluorescence images of CD31, α-SMA, and DAPI staining of the mouse hearts in each group, where CD31 is used as an endothelial cell marker, α-SMA as a smooth muscle cell marker, and DAPI as a marker for cell nuclei. The results showed that compared with the saline group, the number of CD31-positive blood vessels in the tanshinone sodium treatment group, especially in the high-dose group, increased significantly, indicating that tanshinone sodium promotes the formation of new blood vessels by promoting endothelial cell proliferation and migration. In addition, the increase in α-SMA-positive smooth muscle cells further supports the maturation and stability of microvessels. ( Figure 5 )

[0074] 2. Animal Experiments on the Prevention and Treatment of HFpEF-Related Coronary Microvascular Disease with Danshensu Sodium

[0075] 1. Medicines

[0076] Danshensu sodium: provided by Shanghai Qiguang Pharmaceutical Co., Ltd., pale white powder, content>98.0% (HPLC).

[0077] 2. Experimental Animals

[0078] Eight-week-old male C57BL / 6J mice (purchased from the Shanghai SLAC Laboratory Animal Center) weighed approximately 20-22 g and were in good health. The experiment began after one week of adaptive feeding. All animals were housed in a barrier system animal room with a constant temperature and humidity (22±2°C, 50-60% humidity), with a circadian rhythm of 12 hours of light and dark cycle, and were allowed to drink water and eat freely. The control group mice were fed a standard laboratory chow, and the remaining mice were used for model construction. All animal experimental operations followed the animal experimental operation procedures approved by the "Regulations on the Management of Laboratory Animals".

[0079] 3. Construction of HFpEF-related CMVD Animal Model

[0080] A high-fat diet combined with DOCA (deoxycorticosterone acetate) was used to induce CMVD in the HFpEF-related model. Model mice were fed a high-fat, high-cholesterol diet (40% fat and 1.25% cholesterol, purchased from Research Diets) for 16 weeks to induce metabolic disturbances and obesity. At the end of week 16, a sustained-release DOCA pellet (Innovative Research of America, 50 mg / tablet, sustained release for 8 weeks) was implanted subcutaneously under anesthesia (induced and maintained with isoflurane). Simultaneously, 0.9% sodium chloride was administered in drinking water to simulate mild hypertension. Pellets continued to release the pellet until week 24. Body weight and metabolic parameters were monitored biweekly during this period. At the end of week 24, cardiac structure and function, including left ventricular diastolic function (E / A ratio, E / e') and ejection fraction (EF), were assessed using high-frequency ultrasound imaging (VisualSonics Vevo 2100). Coronary microcirculatory function was also assessed using transcoronary flow reserve (CFR).

[0081] 4. Animal Grouping and Drug Intervention

[0082] Mice that were successfully modeled were randomly divided into four groups of eight mice each, with a total experimental period of 24 + 4 = 28 weeks. Starting from the 24th week of modeling (i.e., the eighth week after DOCA implantation), continuous gavage administration was initiated for four weeks. The treatments for each group were as follows:

[0083] Control group (Control): standard diet, no high-fat feeding and DOCA treatment, gavage with normal saline;

[0084] Model group: model mice, gavage with normal saline;

[0085] Low-dose sodium danshensu group (Sodium Danshensu-MD): Model mice were orally administered sodium danshensu 150 mg / kg / day;

[0086] High-dose sodium danshensu group (Sodium Danshensu-HD): Model mice were gavaged with 450 mg / kg / day of sodium danshensu.

[0087] All drugs were administered orally at an equal volume (10 mL / kg) once daily for four weeks. During this period, the mice's mental state, weight changes, and mortality were observed to ensure the safety and effectiveness of the intervention.

[0088] 5. Main testing indicators

[0089] (1) Metabolic related indicators:

[0090] Body weight and heart weight (hearts were removed and weighed after perfusion with saline, after removal of accessory tissues), fasting blood glucose (blood drawn from the tail vein using a OneTouch blood glucose meter), serum insulin levels (ELISA kit, ThermoFisher), blood lipids (total cholesterol and triglycerides, enzymatic colorimetric assay), and noninvasive tail-cuff blood pressure (BP-98A blood pressure measurement system, Softron) were measured before and after oral gavage. All blood samples were collected by retroorbital bleed or cardiac puncture after an 8-hour fast.

[0091] (2) Cardiac microcirculation function test:

[0092] CFR assessment: Coronary blood flow velocity was measured using a VisualSonics high-frequency ultrasound device. The blood flow reserve ratio (coronary flow velocity after stimulation / under basal conditions) was assessed after deep anesthesia induced by 3.0% isoflurane.

[0093] (3) Histopathological examination:

[0094] Heart tissues were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned (5 μm) for HE and Masson staining to assess the degree of myocardial fibrosis and tissue structural integrity;

[0095] (4) Vascular density measurement

[0096] Immunofluorescence staining was used to detect the number of blood vessels labeled with CD31 and α-SMA, and images were acquired using a confocal microscope (Leica SP8) and quantitatively analyzed using ImageJ software;

[0097] (5) Myocardial cell area analysis

[0098] The cross-sectional area of ​​myocardial cells was determined after wheat germ agglutinin (WGA) staining of paraffin sections of mouse hearts. Ten fields of view were randomly selected from each animal for measurement.

[0099] 6. Experimental Results

[0100] (1) Changes in basal metabolic parameters and the effects of drug intervention

[0101] The model group mice had DOCA-induced hypertension, which was effectively reduced by tanshinone sodium (low and high doses). In addition, although fasting blood glucose and insulin levels fluctuated in the model group, no significant changes were observed in the treatment groups after intervention. The low-dose tanshinone sodium group significantly increased HDL levels. At the same time, low and high doses of tanshinone sodium also significantly reduced LDL, triglyceride (TG) and total cholesterol levels, further demonstrating its advantages in improving metabolic status. ( Figure 6 )

[0102] (2) Danshensu sodium alleviates myocardial fibrosis and reduced vascular density

[0103] Danshensu sodium treatment significantly reduced the degree of myocardial fibrosis, especially the high-dose group, which was superior to the low-dose treatment group in histopathological scores. In CD31 and α-SMA immunostaining, Danshensu sodium intervention significantly restored the density of arterioles and capillaries, suggesting that it has the effect of promoting angiogenesis and alleviating microcirculatory remodeling. ( Figure 7 )

[0104] (3) Improve cardiac diastolic function

[0105] The model mice showed a decreased E / A ratio and decreased E wave peak velocity on cardiac ultrasound, indicating significant diastolic dysfunction. After low- and high-dose intervention with tanshinone sodium, all parameters improved significantly, with left ventricular diastolic parameters returning to levels close to those of the control group. The E / A ratio and E wave velocity increased, suggesting that tanshinone sodium can effectively alleviate diastolic dysfunction caused by HFpEF-related CMVD and improve cardiac compliance. Figure 8 )

[0106] (4) Restore coronary reserve blood flow and microcirculatory function

[0107] The CFR of the model mice decreased significantly (CFR < 2), and tanshinone sodium treatment could significantly restore the CFR. Figure 9 )

[0108] 3. Animal Experiments on Danshensu in Preventing and Treating Type 2 Diabetes-Related Coronary Microvascular Disease

[0109] 1. Medicines

[0110] Danshensu sodium: provided by Shanghai Qiguang Pharmaceutical Co., Ltd., pale white powder, content>98.0% (HPLC).

[0111] 2. Experimental Animals

[0112] Fifteen endothelial lineage-tracing mice (Cdh5-Cre+ / - / Rosa26-mTmG+ / +, Shanghai Tongfeng Huiji Biopharmaceutical Technology Co., Ltd.), 6 weeks old and weighing 18-20 g, were obtained by crossbreeding Cdh5-Cre mice (B6-Cdh5tm1(iCre / ERT2) / Bcgen) and Rosa26-mTmG mice (B6.129(Cg)-Gt(ROSA)26Sortm4(ACTB-tdTomato, EGFP)Luo / J). The mice were housed in a room with a temperature of 22-26°C, a humidity of 40%-70%, and a 12-h / day alternating light-dark cycle. Feed and water were available ad libitum.

[0113] 3. Construction of Diabetes-Related Coronary Microvascular Disease Model

[0114] Six-week-old Cdh5-Cre+ / - / Rosa26-mTmG+ / + mice were weighed and injected intraperitoneally with 75 mg / kg tamoxifen for five consecutive days. The mice were stabilized for one week after the last tamoxifen injection. Eight-week-old Cdh5-Cre+ / - / Rosa26-mTmG+ / + mice were weighed and injected intraperitoneally with a single STZ (75 mg / kg) intraperitoneal injection. The mice were then fed a 60% high-fat diet for 16 weeks. At the time of modeling, each group of mice fasted for 12 hours. Blood was collected from the tail vein of each mouse, and blood glucose levels were measured using a Bayer blood glucose meter. A fasting blood glucose level greater than 11 mmol / L was considered a successful model. Successful Cdh5-Cre+ / - / Rosa26-mTmG+ / + mice were randomly divided into three groups of five mice each. Based on the drug's equivalent dose, low- and high-dose groups of Danshensu sodium were established, with oral administration of 150 and 450 mg / kg, respectively. A negative control group was given an equal volume of normal saline. Dosing began at 8:00 AM for four consecutive weeks. The animals were observed for coronary microcirculatory function, cardiac vessel density, and inflammation at these doses.

[0115] Negative control group: gavage with normal saline;

[0116] Low-dose danshensu sodium group: oral administration of danshensu sodium 150 mg / kg / day;

[0117] High-dose sodium tanshinone group: oral administration of sodium tanshinone 450 mg / kg / day.

[0118] 4. Observation indicators

[0119] (1) CFR test

[0120] Coronary flow velocity reserve in mice was assessed using a high-frequency ultrasound imaging system (Vevo 2100 VisualSonics). After chest hair removal, all mice were anesthetized with 2.5% isoflurane. Anesthesia was maintained with 1% isoflurane for 10 minutes. A 40 MHz MS550D transducer was angled to visualize the long axis of the coronary arteries in B-mode. Blood flow direction was observed in Color-Doppler mode. Systolic and diastolic images of resting coronary blood flow velocity were recorded in PW-mode, and diastolic blood flow velocity values ​​were measured. Subsequently, coronary artery dilation was induced with 2.5% isoflurane for 10 minutes, and coronary blood flow velocity was measured under stress as described above. The coronary flow rate (CFR) was calculated for each mouse.

[0121] (2) Autopsy and histopathological examination

[0122] Number of autopsies: all animals scheduled for autopsy;

[0123] Necropsy method: After intraperitoneal injection of pentobarbital (150 mg / kg) for anesthesia, necropsy was performed after cervical dislocation;

[0124] Cardiac tissue fixation and preservation: Open the animal's chest cavity and visually inspect the tissues and organs for obvious abnormalities. Use 10 ml of normal saline to irrigate the heart, then obtain the intact heart tissue, remove the cardiac envelope, and fix the heart tissue with tissue fixative.

[0125] Histopathological examination: Preparation of paraffin sections: After fixing, some tissue was trimmed and sampled, dehydrated in graded alcohol, embedded in paraffin, and sliced ​​using a sliding microtome (LEICARM2135 model) to a thickness of approximately 5 μm. White paraffin slices were obtained using glass slides, and the remaining tissue paraffin-embedded blocks were stored at room temperature. Preparation of frozen sections: The remaining heart tissue was placed in an embedding cassette, OCT was poured into the cassette to completely immerse the tissue, and the tissue was gently placed in liquid nitrogen for 10 seconds. After freezing, it was stored in a -20°C refrigerator; the frozen sections were sliced ​​with a freezing microtome to a thickness of 5 μm; the cut tissue slices were quickly transferred to glass slides, with two slices of tissue attached to each slide; the prepared sections and the remaining tissue embedded blocks were stored in a -20°C refrigerator.

[0126] The paraffin white sections of the hearts of animals in each group were placed in a 70°C oven for 1 hour. After dewaxing and hydration, antigen retrieval, membrane permeabilization, incubation and other processing steps, they were examined under a fluorescence microscope and photographed.

[0127] The frozen sections of the hearts of the animals in each group were covered with polyformaldehyde, broken through the membrane, circled with a histochemical pen, blocked, incubated, stained with nuclei, and sealed. They were then observed and photographed under a specific laser on a fluorescence microscope to collect images.

[0128] (3) Data statistical analysis

[0129] All values ​​are expressed as mean ± standard error. Data were analyzed for normal distribution using the Shapiro-Wilk test. Differences between two groups were analyzed using the Student's t-test. Differences between multiple groups were analyzed using one-way analysis of variance (ANOVA). Data with skewed distributions were analyzed using the Mann-Whitney U-test or Kruskal-Wallis test. Differences were considered statistically significant when the P value was less than 0.05. Statistical analysis and graphing were performed using GraphPad Prism 9.0.

[0130] 5. Experimental Results

[0131] (1) Effect of Danshensu Sodium on CFR in Model Mice

[0132] The CFR of model mice decreased significantly (CFR<2), and oral administration of low-dose (150mg / kg) and high-dose (450mg / kg) tanshinone sodium significantly improved the CFR ( Figure 10 ).

[0133] (2) Pathological examination

[0134] Low-dose (150 mg / kg) and high-dose (450 mg / kg) tanshinone gavage treatment increased cardiac vascular density in mice and reduced macrophage infiltration ( Figure 11 ).

[0135] 6. Conclusion

[0136] Continuous oral administration of 150 mg / kg and 450 mg / kg of sodium danshensu solution to model mice for 4 weeks significantly improved their coronary microcirculation function and cardiac vascular density, and inhibited the infiltration of cardiac macrophages.

Claims

1. Use of danshensu or a pharmaceutically acceptable salt thereof in the preparation of a medicament for preventing, improving or treating atherosclerosis-related coronary microcirculatory diseases, heart failure with preserved ejection fraction (HFpEF)-related coronary microvascular diseases, and diabetes-related coronary microvascular diseases.

2. The use according to claim 1, characterized in that The diabetes is type 2 diabetes.

3. Use of a pharmaceutical composition comprising danshensu or a pharmaceutically acceptable salt thereof in the preparation of a medicament for preventing, improving or treating atherosclerosis-related coronary microcirculatory disease, HFpEF-related coronary microvascular disease, and diabetes-related coronary microvascular disease, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable excipient and / or carrier, and the pharmaceutical composition uses danshensu or a pharmaceutically acceptable salt thereof as an active ingredient.

4. The use according to claim 3, characterized in that The diabetes is type 2 diabetes.

5. The use according to any one of claims 1 to 4, characterized in that The excipients include any one or a combination of at least two of diluents, excipients, fillers, stabilizers, binders, wetting agents, disintegrants, emulsifiers, cosolvents, solubilizers, osmotic pressure regulators, surfactants, coating materials, pH regulators, antioxidants, antibacterial agents, colorants or buffers.

6. The use according to any one of claims 1 to 4, characterized in that The medicine is in the form of a preparation or health food, the preparation is a tablet, granule, capsule, pill, oral liquid or injection, and the health food is a herbal tea, brewed beverage or solid food.

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