Assessment of Tanshinone IIA Derivatives for Cardioprotection in Myocardial Ischemic Injury

• Abstract
• Introduction
• Results
• Discussion
• Materials and Methods
• Materials
• In vitro biological evaluation
• Liver microsome metabolic stability test of I-3
• Myocardial ischemia protective activity test in vivo
• Statistical analysis
• Contributorsʼ Statement
• References

Abstract

Tanshinone ⅡA (TSA), a component of traditional Chinese medicine, effectively protects against myocardial injury. However, its clinical application is limited by poor water solubility and a short half-life. In this study, we report on four TSA derivatives designed and synthesized by our research group. The protective activity against hypoxia-reoxygenation injury in cells was evaluated, and derivative Ⅰ-3 was selected for in vivo experiments to verify its myocardial protective activity in rats with myocardial infarction. The results demonstrated that these four compounds could protect neonatal rat cardiomyocytes from hypoxia-reoxygenation injury. Among the derivatives, Ⅰ-3 showing superior protective effects, we found that Ⅰ-3 has enhanced metabolic stability and an extended half-life. Ⅰ-3 exhibited superior biological activity, effectively reducing the heart infarction area, alleviating myocardial hypertrophy, and enhancing cardiac pumping function. Ⅰ-3 reported in the present work represents a novel and effective derivative of TSA, showing great potential for the treatment of myocardial ischemia (MI).

Introduction

Coronary atherosclerotic heart disease (CHD), also known as ischemic heart disease, is caused by myocardial ischemia (MI) and hypoxia due to coronary atherosclerosis. According to the World Health Organization, ischemic heart disease is currently the leading cause of death globally [1]. It is also the second-leading cause of cardiovascular death in China [2]. MI, the most common symptom in clinical cardiovascular diseases, is directly caused by decreased blood pressure, reduced aortic blood supply, and coronary artery obstruction [3]. Coronary atherosclerosis, changes in blood viscosity, valvular heart disease, and inflammation can also indirectly reduce blood supply to the heart [4]. Restoring blood supply to the heart can enhance patient survival and improve quality of life.

Salvia miltiorrhiza Bunge, also known as red sage or Danshen (Chinese Pinyin name), is a perennial plant in the genus Salvia of the Lamiaceae family. Its roots are highly valued as a “super grade herb (herbs lacking observable toxicity)” in Shennongʼs Herbal Classic of Materia Medica (Shennong Bencao Jing), written during the reigns of the Qin and Han dynasties (221 BC to 220 AD) [5]. Modern research has demonstrated that Salvia miltiorrhiza possesses various pharmacological activities, including anti-inflammatory, antioxidant, and anti-apoptotic properties. Numerous preclinical investigations have demonstrated that it possesses the potential for the treatment of neurodegenerative disorders and cancer [6], [7], [8], [9], [10]. Additionally, its cardiovascular protective efficacy has been most prevalently reported [11], [12], [13]. Salvia miltiorrhiza is a key component of several traditional Chinese medicine formulas, including Danshen Dripping Pills and Shenqi Capsules. It contains over 30 lipophilic compounds with diterpene ketone structures and more than 50 hydrophilic compounds with phenolic acid structures [14]. TSA, the most abundant monomer among the lipophilic compounds in Salvia miltiorrhiza, has been identified as its primary active component in previous studies.

Research on the cardiovascular effects of TSA has received considerable attention from scholars in the medical field, especially in traditional Chinese medicine, both in China and abroad, research by Fu et al. [15] shows that TSA exhibits antioxidant effects both in vivo and in vitro, thereby inhibiting cardiomyocyte apoptosis and providing myocardial protection. Evidence suggests that the beneficial effects of TSA are related to the upregulation of the Bcl-2/Bax ratio and serum superoxide dismutase (SOD) activity, as well as the inhibition of serum malondialdehyde levels. Additionally, research by Gao et al. [16] suggests that TSA can ameliorate doxorubicin-induced myocardial lipid peroxidation. Nitric oxide is an effective vasodilator involved in the regulation of vascular tone. Various studies indicate that TSA can activate endothelial nitric oxide synthase (eNOS), leading to vasodilation and reduced blood pressure [17], [18]. Furthermore, numerous studies have reported the importance of calcium signaling in the regulation of ventricular hypertrophy. TSA has been proven to be a calcium antagonist, significantly modulating calcium ion flux by reducing calcium ion release or blocking L-type calcium channels [19]. These studies collectively demonstrate the significant potential of TSA in the treatment of cardiovascular diseases.

However, TSAʼs clinical use is constrained by its poor water solubility, short half-life, and poor bioavailability [20], [21]. Therefore, we modified the structure of TSA to develop drugs with improved metabolic stability and higher water solubility. We wanted to determine whether these derivatives could provide protection against myocardial ischemic injury, especially in comparison to TSA itself, so we evaluated the biological activity of these derivatives.

The structure of TSA offers numerous derivatization sites. To enhance the metabolic stability and water solubility of TSA, we designed four derivatives: I-3, III-2, 3, and 3a (structures shown in [Fig. 1]) [22], [23]. We tested the biological activity of these derivatives in neonatal rat cardiomyocytes to observe their protective effects on myocardial cell injury induced by hypoxia-reoxygenation. Subsequently, we conducted in vivo experiments on I-3 to investigate its protective effect on myocardial ischemic injury caused by ligation of the left anterior descending branch of the coronary artery in rats.

Results

We evaluated the preventive and therapeutic effects of TSA and its derivatives on hypoxia-reoxygenation injury of neonatal rat cardiomyocytes. [Fig. 2] illustrates the effects of TSA and its derivatives on the viability of neonatal rat cardiomyocytes in a hypoxia-reoxygenation model. The results showed that under normoxic conditions, various concentrations of the drugs had no significant impact on cell viability. However, after 2 hours of hypoxia followed by 4 hours of reoxygenation, cardiomyocyte viability significantly decreased, indicating severe damage caused by hypoxia-reoxygenation. Treatment with TSA and its derivatives markedly improved the viability of reoxygenated cardiomyocytes, with pretreatment demonstrating a more pronounced protective effect compared to treatment alone. Additionally, a concentration of 3 µM was identified as optimal, offering both safety and efficacy. Lower concentrations had little effect, while higher concentrations caused cytotoxicity and reduced cell viability. Our data indicated that Ⅰ-3 demonstrated superior protective effects against hypoxia-reoxygenation injury in cardiomyocytes compared to TSA, with excellent safety and stability. Given the high bioavailability of Ⅰ-3 observed in previous studies, it was selected for subsequent in vivo experiments to further evaluate its protective effects and underlying mechanisms in myocardial infarction.

In vitro experiments demonstrated that I-3 provided the most effective protection against hypoxia-reoxygenation injury in cardiomyocytes, making it the focus of further investigation. The evaluation of its liver microsomal metabolic stability is shown in [Fig. 3]. Results revealed that the half-life of I-3 in rat liver microsomes was 31.29 ± 2.11 minutes, and the clearance rate of liver microsomes CL was 0.02 225 ± 0.0015 mL/min*mg. The half-life of TSA in rat liver microsomes is exceedingly brief, lasting less than two minutes [25], [26], which is significantly shorter than that of Ⅰ-3.

We also evaluated the protective effects of TSA and Ⅰ-3 on myocardial infarction and hypertrophy. In this study, a rat model of myocardial infarction was established by ligation of the left anterior descending artery (LAD), and the effects of TSA (51 mmol/kg) and Ⅰ-3 (34 mmol/kg) were evaluated. The changes in myocardial infarction size were assessed using TTC staining, as shown in [Fig. 4]. Compared to the sham group, the myocardial infarction group exhibited a significant white infarct area. In contrast, the infarct area was significantly reduced in the TSA and Ⅰ-3 treatment groups, indicating that both drugs effectively reduce myocardial infarction size.

Additionally, myocardial hypertrophy was assessed by measuring the heart-to-body weight ratio (mg/g) and the heart-to-tibial length ratio (mg/cm), as shown in [Fig. 5]. The heart weight of rats in the myocardial infarction group was significantly higher than that of the sham group, suggesting the presence of myocardial hypertrophy. However, the heart weights in the TSA and Ⅰ-3 treatment groups were significantly reduced, indicating that both drugs effectively mitigate myocardial hypertrophy.

In conclusion, TSA and Ⅰ-3 not only significantly reduce the size of myocardial infarction but also improve myocardial hypertrophy, demonstrating their potential as therapeutic agents for pathological changes associated with myocardial infarction.

Then we investigated the protective effects of TSA and Ⅰ-3 on myocardial enzyme activity changes following myocardial infarction. This study evaluated the changes in plasma myocardial enzyme activity (CK, CKI/CK-MB, and LDH) in the sham group, myocardial infarction group, TSA treatment group (51 mmol/kg), and Ⅰ-3 treatment group (34 mmol/kg) at 2, 3, and 24 hours post-myocardial infarction ([Fig. 6]). Changes in plasma myocardial enzyme activity reflect the extent of cardiac injury to some degree [27].

The results showed that myocardial enzyme activity changes were unstable within the first 2 hours post-surgery. However, at 3 hours, the myocardial infarction group exhibited a significant increase in plasma myocardial enzyme activity, indicating aggravated cardiac injury. In contrast, the TSA and Ⅰ-3 treatment groups significantly reduced the elevated plasma myocardial enzyme activity compared to the myocardial infarction group, suggesting that both drugs effectively alleviate cardiac injury caused by myocardial infarction. By 24 hours post-surgery, plasma myocardial enzyme activity gradually returned to normal levels.

In summary, TSA and Ⅰ-3 provide early stage protection against cardiac injury following myocardial infarction, demonstrating their potential therapeutic value.

As for the protective effects of TSA and Ⅰ-3 on cardiac function in rats with myocardial infarction, cardiac catheterization is a classic method for evaluating cardiac pumping function by assessing hemodynamic parameters. This study compared the changes in hemodynamic values among the sham group, myocardial infarction group, TSA treatment group (51 mmol/kg), and Ⅰ-3 treatment group (34 mmol/kg) ([Fig. 7]). The maximum rate of left ventricular pressure rise and left ventricular systolic pressure (LVSP) reflect left ventricular systolic function, while left ventricular end-diastolic pressure (LVEDP) and the maximum rate of left ventricular pressure decline reflect left ventricular diastolic function [28].

The results showed that, compared with the sham group, the hemodynamic parameters in the myocardial infarction group were significantly reduced 14 days post-modeling, indicating severe impairment of left ventricular systolic and diastolic functions due to coronary artery ligation. In contrast, both the TSA and Ⅰ-3 treatment groups exhibited significant improvement in hemodynamic parameters, suggesting that these drugs effectively enhanced left ventricular systolic and diastolic functions, partially restoring cardiac pumping ability.

In summary, TSA and Ⅰ-3 demonstrate a significant protective effect against myocardial infarction-induced cardiac dysfunction, highlighting their potential therapeutic value.

Discussion

This study employed a hypoxia-reoxygenation model using neonatal rat cardiomyocytes to compare the cardioprotective activities of four derivatives of TSA. The results indicated that all four derivatives alleviated hypoxia-reoxygenation injury in neonatal rat cardiomyocytes to varying degrees. Notably, derivative I-3 exhibited a significantly greater protective effect than TSA. Consequently, we assessed the metabolic stability of I-3 and conducted in vivo experiments. Our findings indicate that in rat liver microsome stability tests, I-3 demonstrates a longer half-life and enhanced metabolic stability. Furthermore, I-3 provides superior cardiac protection in a rat coronary artery ligation-induced MI model compared to TSA. It mitigates myocardial ischemic injury by ameliorating myocardial hypertrophy, reducing the infarct area, and restoring cardiac pump function. Therefore, we propose that modifying the structure of TSA to enhance its in vivo metabolic stability may improve its efficacy in treating MI. This provides valuable insights for the design and development of more effective drugs for MI.

Salvia miltiorrhiza, due to its multi-target properties, exhibits therapeutic effects on various diseases, making it a prominent subject of research. Numerous studies have focused on the structural modification of TSA. Derivatives developed through various design approaches exhibit unique characteristics. Consequently, their effects on different diseases vary. We investigated various derivatives of TSA for treating MI. Increasing water solubility alone may not significantly enhance the myocardial protective effect of TSA. For instance, derivative 3a, which uses amino acids as spacers and is coupled with polyethylene glycol (PEG), exhibited excellent water solubility and drug release capability. However, our experimental studies showed that its performance, both in vivo and in vitro, did not surpass that of TSA. Instead, despite having inferior water solubility compared to 3a, I-3 with a longer half-life and higher metabolic stability demonstrated better protection against myocardial ischemic injury. This indicates that improving the metabolic stability of TSA may be a more suitable design strategy for treating myocardial ischemic injury. We did not further investigate the mechanisms through which I-3 protects against myocardial ischemic injury. If certain targets can be validated to play prominent roles in this process, we can speculate that these targets are crucial for treating myocardial ischemic injury. Furthermore, new derivatives can be designed based on these targets to evaluate their potential for better therapeutic effects. This provides a direction for the future design and research of MI treatment drugs.

In summary, this paper confirms the efficacy of four TSA derivatives that were designed and synthesized by our research group for protecting against myocardial ischemia, with 3 – 2 demonstrating exceptional performance, favorable metabolic stability, and an extended half-life. Furthermore, in vivo studies suggest that its cardioprotective effects are superior to those of TSA, providing a foundation for the structural modification of TSA in the context of treating ischemic heart disease.

Materials and Methods

Materials

The 200 – 240 g male Sprague–Dawley rats were purchased from Hua Chuang Sino Medical Technology Co., Ltd (Purchase License No.: SCXK (Su) 2018 – 0019 Sales License No.: SCXK (Su) 2020 – 0009); neonatal Sprague–Dawley rats (1 – 3 days old), male or female, were purchased from Hua Chuang Sino Medical Technology Co., Ltd (Purchase License No.: SYXK (Su) 2018 – 0019; Sales License No.: SCXK (Su) 2020 – 0009). The animal experiments in this study were conducted at the Animal Experiment Center of the China Pharmaceutical University and approved by the Animal Ethical Committee of the China Pharmaceutical University (date of approval: December 19th, 2024; approval number: 2024 – 12 – 089).

Tanshinone ⅡA was purchased from Xiʼan Shen Nong Biotechnology Co., Ltd, with the purity over 98%; DMEM high-glucose/low-glucose medium was purchased from Bio-Channel Biotechnology Co., Ltd; type II collagenase was purchased from Sangon Biotech Co., Ltd; trypsin was purchased from Ranjeck Technology Co., Ltd; extra-grade fetal bovine serum was purchased from Clark Bioscience; superior fetal bovine serum (Every Green) was purchased from Tian Hang Biotechnology Co., LTD; DMSO was purchased from Solarbio Science & Technology Co., Ltd; CCK-8 (Cell Counting Kit-8) was purchased from Vazyme Biotech Co., LTD; 2,3,5-triphenyltetrazolium chloride was purchased from Servicebio Technology Co., LTD; heparin sodium was purchased from Sangon Biotech Co., Ltd; pentobarbital sodium was purchased from Merck.

In vitro biological evaluation

Dulbeccoʼs modified Eagle medium (DMEM) high-glucose, low-glucose, and no-glucose media were prepared and configured; 1% trypsin and 1% type II collagenase were weighed and dissolved in phosphate-buffered saline (PBS) solution. After complete dissolution, the enzyme solution was filtered through a 0.22 µm microporous filter membrane within a clean bench for subsequent utilization.

Neonatal rats (1 – 3 days old) were sterilized by soaking in 75% alcohol for 30 seconds, then transferred to a clean bench where the skin tissue between the armpit and diaphragm was cut with bent scissors. The heart was removed with bent tweezers and placed in a petri dish containing PBS. Excess lung tissue and blood vessels were removed, and the heart was carefully cleaned three times. The myocardial tissue was cut into fragments smaller than 1 mm³ in a small beaker. The myocardial fragments were quickly transferred to a sterile serum vial, combined with the prepared enzyme digestion solution, and digested in a water bath at 37℃ for 13 minutes. The slightly cloudy digestive liquid was then transferred to a 10 mL centrifuge tube containing an equal volume of low-glucose medium. It was thoroughly mixed to terminate the digestion process and preserve the cells. Fresh digestive solution was added to the serum vial and digested in a water bath at 37℃ for 13 minutes. This process was repeated 5 – 7 times. All centrifuge tubes were wrapped with sealing film and centrifuged at 145 × g for 10 minutes. The supernatant was removed, and low-glucose medium was added to resuspend the cell pellet. The cells were transferred to a culture dish after filtration through an 80-mesh sieve, and the culture dish was placed in a carbon dioxide incubator for 90 minutes. During this period, most fibroblasts adhered to the dish and grew. After the culture period, the old medium in the petri dish was transferred to a 10 mL centrifuge tube and centrifuged at 145 × g for 5 minutes. The supernatant was removed, and high-glucose medium was added to resuspend the cell pellet. A 10 µL sample was taken for cell counting, and the remaining cells were inoculated in a petri dish.

Cultured neonatal rat cardiomyocytes were randomly divided into five groups: control, sham, hypoxia-reoxygenation model, pretreatment, and treatment. The control group was cultured in a carbon dioxide incubator; the sham group was cultured in a carbon dioxide incubator after the drugs were added to the cell culture medium; the hypoxia-reoxygenation model, pretreatment, and treatment groups were cultured in a hypoxic incubator (N₂ 93%, O₂ 2%, CO₂ 5%) for 2 hours, followed by reoxygenation in a carbon dioxide incubator for 4 hours. In the pretreatment group, drugs dissolved in dimethyl sulfoxide (DMSO) were added before hypoxic culture at final concentrations of 0.3 µM, 3 µM, and 30 µM; in the treatment, drugs were added after hypoxia at the final concentrations.

The viability of neonatal rat cardiomyocytes was measured using a Cell Counting Kit-8 (CCK-8) assay. The cells were inoculated in a 96-well plate (about 5000 cells per well). After hypoxia-reoxygenation and drug treatment, 10 µL of CCK-8 reagent was added to each well of a 96-well plate, gently tapped the edge of the plate to mix, and incubated in a carbon dioxide incubator for 1.5 hours. The absorbance at 450 nm was measured using a microplate reader. Cell viability was calculated as (experimental group OD value – blank group OD value)/(control group OD value – blank group OD value) × 100%.

Liver microsome metabolic stability test of I-3

Three parallel experimental groups and one negative control group were established. In a 37 °C incubator, each system sequentially received 100 µL of 0.1 M PBS buffer, 50 µL of MgCl₂ solution (final concentration of 5 mM), 20 µL of prepared compound solution (final concentration of 1.5 µM), and 10 µL of rat liver microsomes (8 mg/mL, final concentration of 0.4 mg/mL), resulting in a final reaction volume of 200 µL. The reaction mixture was blended and incubated at 37 °C for 5 minutes.

After 5 minutes of incubation, 20 µL of NADPH (10 mM) was added to the bottom of the tube using a pipette tip to initiate the reaction (the negative group did not receive NADPH), and samples of 20 µL were taken at seven time points: 0, 5, 15, 30, 45, 60, and 90 minutes. Each sample was mixed with 250 µL of chromatographic methanol containing 60 ng/ml internal standard to terminate the reaction, while the entire system was maintained at 37 °C throughout the experiment.

Proteins were precipitated, and the reaction was centrifuged at 13 680 × g for 10 minutes. The supernatant was collected for LC-MS/MS analysis. Three replicates were prepared in parallel.

Negative control group: no NADPH was added; only PBS buffer was included to assess non-oxidative metabolism.

Establishment and preparation of the standard curve: the experimental system was consistent with the aforementioned setup, using inactivated liver microsomes (inactivation method: liver microsomes were treated in water at 100 °C or 60 °C for 1 hour). The final concentration range of the compounds was 1 ng/mL to 1000 ng/mL (the detection range of LC-MS/MS), with a gradient setup based on this concentration (9 to 10 samples).

Data processing: the concentration of the compound at the 0-minute sampling point after the reaction started was set as 100%, and the concentrations at other incubation points were converted to percentage remaining. The natural logarithm of the percentage remaining at each time point was subjected to linear regression against incubation time to calculate the slope K. Using the formula t_1/2 = − 0.693/K, the in vitro half-life can be calculated. The clearance rate of liver microsomes CL (mL/min*mg) is 0.693/t_1/2.

Myocardial ischemia protective activity test in vivo

After adaptive feeding, Sprague–Dawley male rats were fasted from solids and liquids for 2 hours and weighed before the experiment. The rats were anesthetized with an intraperitoneal injection of 1% pentobarbital sodium at a dose of 60.4 mmol/kg, then the BL-420 s biological function experiment operating system was connected to record lead II electrocardiograms. The neck and chest were alternately disinfected with iodine and alcohol. A longitudinal incision of about 1 cm was made in the midline of the neck using surgical scissors. The superficial skin fascia, subfascial tissue, and anterior neck muscle were separated layer by layer. The trachea was then exposed, a small opening was made in the trachea, and a catheter was inserted to a suitable depth. The catheter was connected to a ventilator so that the chest rose and fell in sync with the ventilator frequency. A longitudinal incision about 1 cm long was made at the left margin of the sternum, and the pectoralis major and serratus anterior muscles were bluntly separated layer by layer. The intercostal muscles were exposed at the third and fourth intercostal spaces. The intercostal muscles were bluntly separated from the thoracic cavity with hemostatic forceps in the direction of the intercostal space. The ribs were opened with a chest expander, the pericardium was removed with tweezers, and the pericardium and lung tissues near the heart were separated with wet saline cotton. The left coronary artery was located, and the myocardial tissue containing blood vessels was ligated with 6 – 0 surgical sutures. After stabilizing for 2 – 5 minutes, darkening of the tissue around the ligated myocardium and elevation of the ST segment in the electrocardiogram (ECG) were observed. In the sham group, a 6 – 0 suture was passed through the left anterior descending branch without ligation, with the other steps identical to the coronary ligation group. The chest was then closed in layers. The ribs were sutured with a 4 – 0 surgical suture, air was removed from the chest with a syringe, muscles and skin were sutured, and incisions were disinfected. Absorbent cotton was used to clean up tissue exudation around the trachea. The tracheal intubation was retained, the anterior tracheal muscle and skin were sutured and the intubation fixed, the neck incision was disinfected, and the ventilator was removed. Special attention was paid to ensure the rats could breathe autonomously, and they were kept warm until recovery from anesthesia. The surviving rats were randomly divided into a model group and a drug treatment group. The drug treatment group received an intraperitoneal injection of TSA at 51 mmol/kg/day and I-3 at 34 mmol/kg/day for 14 days post-modeling. The body weight, tibial length, and heart weight of the rats were recorded, and the ratios of heart weight to body weight (mg/g) and heart weight to tibial length (mg/cm) were calculated.

The rat heart was removed, cleaned with PBS, and then frozen at − 20℃ for easier slicing. The frozen heart was cut into five pieces with a blade, soaked in 2% TTC solution, and stained at 37℃ in the dark. After uniform staining, photographs were taken, and the proportion of the infarct area to the left ventricle was analyzed.

At 2, 3, and 24 hours after coronary ligation, 1.5 mL of blood was collected from the ratsʼ eye sockets and centrifuged at 625 × g for 5 minutes to collect the serum. The serum was then removed, and the working reagent (kit: Redu/Changchun Huili) was prepared. The corresponding parameters were set on the automatic biochemical instrument, the sample was loaded, and the instrument automatically performed the measurements.

Fourteen days after coronary ligation, the rats were anesthetized with an intraperitoneal injection of 1% pentobarbital sodium at a dose of 60.4 mmol/kg, and hemodynamic parameters were determined. Adjust the three-way valve of the pressure transducer to the external interface, insert a 2.5 mL syringe, and inject normal saline into the pipeline, ensuring no air is present. Then close the three-way valve. Open the valve again and replace the saline with 1% heparin sodium solution, ensuring no bubbles are present. After anesthesia, the rats were fixed in a supine position on the operating table. The neck skin was disinfected with iodophor, then incised. Tweezers and hemostatic forceps were used to bluntly separate the muscles, locate the right common carotid artery, and separate it with a glass probe. The distal end of the carotid artery was ligated with thread, the proximal end clamped with an artery clamp, and heparin sodium solution was applied. An inverted V-shaped incision was gently made with ophthalmic scissors. A PE50 catheter filled with heparin was inserted into the carotid artery, another suture was tied with a single knot, and the artery clamp was released. The waveform changes were observed as the catheter was advanced toward the ventricle. An obvious sense of perforation was felt when the catheter passed through the aortic valve. At this point, the waveform changed from a carotid pressure wave to a broad ventricular wave, and the sutures near the heart were tightened. After stabilizing for 5 minutes, left ventricular systolic pressure (LVSP), left ventricular diastolic pressure (LVDP), left ventricular end-diastolic pressure (LVEDP), left ventricular average pressure (LVAP), the maximum rise rate of left ventricular pressure (+ dp/dtmax), and the maximum fall rate of left ventricular pressure (-dp/dtmax) were recorded.

Statistical analysis

SPSS Statistics 22.0 (IBM), GraphPad Prism 7.0, and Origin 2017 software were used for statistical analysis and data visualization. Data were presented as mean ± standard deviation (SD) from triplicate experiments performed in parallel unless otherwise indicated. The normal distribution and variance homogeneity were measured first. One-way analysis of variance (ANOVA) was applied to the results in compliance with normal distribution and variance homogeneity, followed by Tukeyʼs multiple comparisons test. Otherwise, the rank-sum test was applied. Differences with p < 0.05 were considered statistically significant.

Contributorsʼ Statement

Data collection: Z. W. Wu, Y. Xu, Z. L. Zhang, X. M. Guo; design of the study: Y. Q. Tang, J. X. Wang, X. M. Guo, Z. L. Zhang; statistical analysis: Z. W. Wu, Y. Xu, J. X. Wang, Z. L. Zhang; analysis and interpretation of the data: Z. W. Wu, Y. Xu, Y. Q. Tang, J. X. Wang, X. M. Guo: drafting the manuscript: Z. W. Wu, Y. Xu, Y. Q. Tang, Z. L. Zhang; critical revision of the manuscript: Z. W. Wu, Y. Q. Tang, J. X. Wang, X. M. Guo

Conflict of Interest

The authors declare that they have no conflict of interest.

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• 26 Bi HC. The pharmacokinetic study of tanshinone ⅡA in rats and the involved mechanisms. [Dissertation]. Guangdong: Sun Yat-sen University; 2007
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• 27 Lott JA, Stang JM. Serum enzymes and isoenzymes in the diagnosis and differential diagnosis of myocardial ischemia and necrosis. Clin Chem 1980; 26: 1241-1250
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• 28 Bugge-Asperheim B, Kjekshus J. Left ventricular pressure and maximum rate of pressure rise as determinants of myocardial oxygen consumption during hemorrhagic hypotension in dogs. Acta Physiol Scand 1970; 78: 174-183
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Correspondence

Publication History

Received: 23 October 2024

Accepted after revision: 24 March 2025

Accepted Manuscript online:
24 March 2025

Article published online:
25 April 2025

© 2025. Thieme. All rights reserved.

Georg Thieme Verlag KG
Oswald-Hesse-Straße 50, 70469 Stuttgart, Germany

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