1. Introduction
Myocardial ischemia is characterized by insufficient coronary artery blood supply to meet the oxygen demand of the myocardium [
1]. Coronary artery spasm and atherosclerosis from various sources, as well as cardiomyocyte hypertrophy and cardiomyopathy, are the main causes of myocardial ischemia and hypoxia [
2]. Ischemic heart disease is one of the leading causes of morbidity and mortality around the world. According to the
Global burden of disease study 2019, there were 197 million patients with ischemic heart disease worldwide in 2019 [
3]. Thus, this disease is a serious, life-threatening, and financial burden on humanity.
The relaxation and contraction responses of healthy blood vessels are prerequisites for maintaining the blood supply to the myocardium. Non-obstructive coronary artery disease, dysregulated dilation response of coronary microvessels, coronary vasomotor disorders, extravascular compressive forces, and coronary microvascular dysfunction, which are closely related to coronary microvascular dysfunction, have been increasingly recognized as important contributors to the signs and symptoms of chronic and acute myocardial ischemia [
4], [
5]. High reactivity of vascular smooth muscle cells (VSMCs) is the main cause of coronary artery dysfunction [
6]. However, the treatment of myocardial ischemia is usually directed toward the occlusive coronary arteries, ignoring the importance of coronary artery motion disorders and coronary microvascular dysfunction [
7]. Therefore, it is necessary to research and develop drugs to alleviate VSMC hyper-reactivity.
Buxu Tongyu (BXTY), a traditional Chinese medicine (TCM) compound preparation that has been clinically used for many years, contains Hongshen, Huangqi, Ciwujia, Chishao, Danshen, and Guizhi. According to the package insert, BXTY is used for the treatment of coronary heart disease and atherosclerosis caused by qi deficiency and blood stasis. BXTY also benefits qi complement deficiency and promotes blood circulation to remove meridian obstruction. However, the underlying mechanism of BXTY is unclear. It is crucial to clarify the action mechanism of traditional drugs with modern language in order to communicate between traditional medicine and modern medical systems, and promote drug research and development. This study aims to confirm the pharmacological effect of BXTY on myocardial ischemia and to clarify the underlying mechanisms.
2. Materials and methods
2.1. Chemicals, reagents, and samples
BXTY extract was obtained from Sunflower Pharmaceutical Group Co., Ltd. (China). The reference compounds paeoniflorin, protocatechuic acid, salvianolic acid A, and salvianolic acid B were purchased from the National Institutes for Food and Drug Control (China). Chromatographic-grade methanol was purchased from Duksan Pure Chemicals Co., Ltd. (Republic of Korea). Mass-spectrometric-grade methanol was purchased from Thermo Fisher Scientific (China) Co., Ltd. Chromatographic-grade phosphoric acid was supplied by Tianjin Fuyu Fine Chemical Co., Ltd. (China). Distilled and deionized water were used for the preparation of samples and solutions. Pituitrin was purchased from Nanjing Xinbai Pharmaceutical Co., Ltd. (China). Nitroglycerin was purchased from Guangzhou Baiyunshan Mingxing Pharmaceutical Co., Ltd. (China). A 3′,5′-cyclic guanosine monophosphate (cGMP) activity assay kit was purchased from Elabscience Biotechnology Co., Ltd. (China). Antibodies against soluble guanylate cyclase (sGC)-β1 and protein kinase G (PKG)-1 were purchased from Sigma-Aldrich (USA) and Cell Signaling Technology (USA), respectively. NS 2028, ODQ, and KT 5823 were purchased from Beyotime Biotechnology (China). Phenylephrine (PE) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. (China). A bicinchoninic acid protein assay reagent (BCA) kit was purchased from Beyotime Biotechnology. Tris-buffered saline (TBS; NaCl 16.0 g, Tris-HCl 4.841 g, dissolved in 2000 mL deionized water) buffer powder was purchased from Boster Biological Technology Co., Ltd. (China).
2.2. Standards and samples preparation
2.2.1. Standards
BXTY powder was accurately weighed and prepared, with the reference solution containing 22 μg·mL−1 of protocatechuic acid, 350 μg·mL−1 of paeoniflorin, 40.5 μg·mL−1 of salvianolic acid A, and 150 μg·mL−1 of salvianolic acid B. The stock solutions were stored at 4 °C and brought to room temperature before use.
2.2.2. Samples
After accurate weighing, 5 g of BXTY was placed in a 50 mL conical flask with a plug, and 20 mL of 70% methanol was added. The drug preparation was shaken well, ultrasonicated for 15 min, and cooled to room temperature, followed by centrifugation for 3 min. The supernatant was divided into two parts, one of which was taken for high-performance liquid chromatography with diode-array detection (HPLC-DAD) and was filtered through a 0.22 μm membrane, with the filtrate being added to the automatic injection flask. The other part of the supernatant was used for ultra performance liquid chromatography-quadrupole-time of flight-tandem mass spectrometry (UPLC-Q-TOF-MSE) analysis, which was first diluted to double volume and filtered through a 0.22 μm membrane. The filtrate was passed through a solid-phase extraction (SPE) column (methanol 5 mL activated SPE column, water balance 5 mL column), and 3 mL of the filtrate was loaded into the sample. After the filtrate was collected, it was kept in a 5 mL volumetric flask, and an appropriate amount of liquid was filtrated through a 0.22 μm membrane into the automatic injection flask.
After 7 d of adaptive feeding and 12 h of fasting, blood was collected at 0, 1, 3, and 5 h after BXTY intragastric administration. Blood samples were collected from the ophthalmic venous plexus into Eppendorf tubes, and 0.6 mL blood samples were collected at each time point. Blood samples were centrifuged at 3000 r·min−1 for 10 min at 4 °C, and 50 μL of supernatant was taken. Next, 5 μL of ascorbic acid solution and 5 μL of formic acid were added to the 50 μL of supernatant and mixed evenly; then, 300 μL of ice methanol was added and the mixture was vortexed for 1 min and centrifuged for 5 min at 12 000 r·min−1. The supernatant was passed through 0.22 μm filter membrane into the automatic injection flask.
2.3. HPLC-DAD and UPLC-Q-TOF-MSE analysis
A Waters 2690 Series liquid chromatography (LC) system (USA) equipped with a 2996 DAD was employed for the HPLC-DAD analysis, and the acquisition wavelength of the DAD detector was set in the range of 190-600 nm. The chromatographic separation of analytes was performed on a Kromasil C18 (4.6 mm × 250 mm, 5 μm). For chromatographic separation, a gradient elution of methanol and 0.3% phosphoric acid in water was used. The gradient elution of the mobile phase was as follows: 0-33 min with 2% methanol; 33-38 min with 2%-22% methanol; 38-65 min with 22% methanol; 65-120 min with 22%-50% methanol; and 120-125 min with 50%-98% methanol. The gradient elution was used at a flow rate of 1 mL·min−1, the column temperature was maintained at 30 °C, and 5 μL of the sample was injected. The wavelength of the DAD detector ranged from 190 to 600 nm. For the UPLC-Q-TOF-MSE measurements, the analysis was performed using a Synapt G2-Si UPLC-Q-TOF-MSE system (Waters). The UPLC separation conditions are given below. An Acquity UPLC BEH C18 column (2.1 mm × 100 mm, 1.7 μm; Waters) was used with an injection volume of 3 μL. The flow rate and column temperature were maintained at 0.3 mL·min−1 and 30 °C, respectively. The mobile phases consisted of methanol and 0.1% formic acid in water. The standard elution program conditions were as follows: 0-1.5 min with 2% methanol; 1.5-11.0 min with 2%-40% methanol; and 11.0-13.5 min with 40%-98% methanol. UPLC-Q-TOF-MSE acquisitions were performed in full information MSE mode with MS analysis by using positive- and negative-ion modes equipped with electrospray ionization (ESI). The m/z of leucine-enkephalin (ESI+, 556.2771; ESI-, 554.2615) was used as a standard for quality determination. The following final MS conditions were used in the positive- and negative-ion detection modes: capillary voltage, 2.5 kV; source temperature, 100 °C; desolvation temperature, 250 °C; low collision energy for precursors, 0 V; high energy for fragment ions, 40 V; cone gas flow, 6.5 L·h−1; and desolvation gas flow, 800 L·h−1. In total, 50-1200 m/z of mass spectrum were collected and analyzed. Data on the chemical compounds isolated from six individual herbs in BXTY were collected and sorted using retrieval databases, including the China National Knowledge Infrastructure (CNKI), PubMed, Web of Science, and ChemSpider. UniFi software was used to generate a self-building library of chemical compounds, including the compound name, molecular formula, and chemical structure, which was then used to process and analyze the MS data. Fragment information was automatically matched, with a minimum peak area of 200 for two-dimensional (2D) peak detection; the peak intensity of high energy over 100 counts and the peak intensity of low energy over 500 counts were selected as the parameters for three-dimensional (3D) peak detection. Some fragments of the structure of the compounds predicted by the UniFi software were generated, and 10 parts per million (ppm) of error for identified compounds was allowed. Our study selected positive adducts including H+, Na+, and NH4+ and negative adducts including HCOO- and H-. Cross-adduct combinations were permitted.
2.4. Animals
Kunming (KM) mice (male, 20-30 g, specific pathogen free (SPF) level) and Sprague-Dawley (SD) rats (male, 150-200 g, SPF level) were purchased from the Laboratory Animal Center of the Second Affiliated Hospital of Harbin Medical University, China. The animals were housed at (23 ± 2) °C in a room with (55% ± 5%) humidity, maintained on a 12 h light/dark cycle, and provided with free access to food and water.
2.5. Mouse acute myocardial ischemia model
Animal experiments were carried out in accordance with the Declaration of Helsinki for animal experiments. After one week of adaptive feeding, the KM mice were randomly divided into seven groups by weight, including the control group, acute myocardial ischemia model group, nitroglycerin group, and four BXTY treatment groups (BXTY-0.5 g·kg−1·d-1, BXTY-2.0 g·kg−1·d-1, BXTY-8.0 g·kg−1·d-1, and BXTY-16.0 g·kg−1·d-1). The animals in the BXTY groups were orally treated with 0.5, 2.0, 8.0, or 16.0 g·kg−1·d-1 for ten consecutive days. The control, model, and nitroglycerin groups were treated with the same volume of deionized water. Ten days later, a myocardial ischemia model was created in the mice of all groups except the control group via the intraperitoneal injection of pituitrin. Nitroglycerin (0.002 mg·g−1) was injected immediately after the intraperitoneal injection of pituitrin to the mice in the nitroglycerin group. Electrocardiograms (ECGs) were recorded by means of a BL420 Biological and Functional Experimental System for 10 min.
2.6. Vasomotor responses
Vasomotor responses were detected using a Multi Myograph System-DMT 620 M (Danish Myo Technology A/S, Denmark). The superior mesenteric arteries isolated from SD rats were placed in a cold and oxygenated physiological saline solution (PSS; 130 mmol·L-1 NaCl, 4.7 mmol·L-1 KCl, 1.18 mmol·L-1 KH2PH4, 1.17 mmol·L-1 MgSO4·7H2O, 14.9 mmol·L-1 NaHCO3, 5.5 mmol·L-1 glucose, 0.026 mmol·L-1 ethylene diamine tetraacetic acid (EDTA), and 1.16 mmol·L-1 CaCl2). The connective tissues around the blood vessels were removed, and the blood vessels were cut into pieces 2-3 mm in length. The isolated arterial rings were fixed by two mounting pins. The PSS in the myograph chamber was aerated with 95% O2 and 5% CO2 and heated to 37 °C. The optimal initial microvascular tension was determined by measuring the passive length-tension relationship through a vascular standardization procedure. A fully relaxed ring was considered to have a transmural pressure of 13.3 kPa. Next, endothelial integrity was validated by inducing vessel pre-constriction with 1 µmol·L-1 PE followed by relaxation with 1 µmol·L-1 carbachol (CCh). Rings with more than 60% relaxation were considered to be endothelium-intact, and those with less than 30% were considered to be endothelium-denuded.
2.7. Cell culture and treatment
Mouse aortic vascular smooth muscle cells (MOVAs) were purchased from BeNa Culture Collection (China). Cells were cultured in Dulbecco’s modified Eagle medium (DMEM) with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin at 37 °C in humidified air containing 5% CO2. After reaching 80%-90% confluence, the cells were incubated with 10 µg·mL−1 BXTY extract for 3, 6, and 9 min or nitroglycerin for 3 min, respectively, with or without 20 µmol·L-1 NS 2028, 100 nmol·L-1 ODQ, or 2 µmol·L-1 KT 5823.
2.8. Determination of cGMP release
The concentration of cGMP released from MOVAs was detected by means of a cGMP enzyme-linked immunosorbent assay (ELISA) kit according to the manufacturer’s instructions. cGMP concentrations were normalized to the protein concentrations.
2.9. Western blot
Protein expressions were semi-quantified using Western blot analysis. Cell samples were lysed in radio immunoprecipitation assay (RIPA) buffer containing protease inhibitor. Protein concentration was quantified using a BCA kit. A 35 µg protein sample extracted from each treatment group was loaded and separated on 10% sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto nitrocellulose membranes. After being blocked in 5% skim milk for 2.5 h, a nitrocellulose membrane was incubated with sGC-β1 and PKG-1 primary antibodies at 4 °C overnight. Next, it was incubated with horseradish peroxidase (HRP)-conjugated secondary antibody at room temperature for 1 h in the dark. Signals were captured using an infrared fluorescence scanning system. The optical density integral value of the protein bands was analyzed by Odyssey 1.3 software.
2.10. Statistical analysis
All results were performed at least three times and are presented as the mean ± standard error of mean (SEM). Statistical significance was estimated by a paired and unpaired Student’s t test for a comparison of the two groups (*P < 0.05, **P < 0.01, and ***P < 0.001).
3. Results
3.1. Determination of the multi-component content of BXTY
We delved into the composition of BXTY by employing the HPLC-DAD technique.
Fig. 1(a) shows the chemical structures of paeoniflorin, salvianolic acid A, protocatechuic acid, and salvianolic acid B.
Fig. 1(b) shows a comparison of the chromatographic peaks of the reference substance and those of the BXTY sample solutions. The results suggest that paeoniflorin is the richest compound in the BXTY extracts (607.21 µg·mL
−1 in total in a BXTY), followed by salvianolic acid B (176.74 µg·mL
−1 in total), salvianolic acid A (38.6 µg·mL
−1 in total), and protocatechuic acid (8.48 µg·mL
−1 in total).
In this study, a combination of UPLC-Q-TOF-MS
E and the UniFi natural product analysis platform was used to identify the chemical components and transmigration components of BXTY. Data was imported into the UniFi analysis platform for automatic matching with the self-built database. Manual checking was also conducted to determine whether the cracking process conformed to the cracking law of the compound. A total of 106 compounds were identified in BXTY, 95 of which entered the blood as prototypes (Table S1 in Appendix A). Base peak intensity (BPI) chromatograms of BXTY and the solvent were acquired in positive- and negative-ion modes (
Figs. 1(c)-(f)). Total ion current (TIC) chromatograms of the transmigration components from BXTY were acquired in positive- and negative-ion modes (
Figs. 1(g) and (h)). We speculated that most of the components in BXTY appeared in the blood as prototypes. Therefore, we used BXTY extracts to conduct the subsequent experiments.
3.2. BXTY alleviates acute myocardial ischemia induced by pituitrin in mice
The main clinical indication of BXTY is coronary heart disease. It has been reported that the main components of BXTY, such as paeoniflorin, salvianolic acid B, salvianolic acid A, and protocatechuic acid, have a protective effect on coronary heart disease [
8], [
9], [
10], [
11]. Here, a mouse model of acute myocardial ischemia induced by pituitrin was used to evaluate the pharmacological effects of BXTY. BXTY was administered by gavage once a day for ten consecutive days. Acute myocardial ischemia was induced by the intraperitoneal injection of pituitrin in mice.
Fig. 2(a) is the representative ECG traces after intraperitoneal injection of pituitrin (0.15 U·g
−1) for 10 min. The amplitude of the T wave in the model mice dramatically increased compared with the control counterparts (
Fig. 2(b)), and the heart rate of the model mice dramatically decreased (
Fig. 2(c)), in comparison with the control mice. These findings indicated the successful establishment of a mouse model of acute myocardial ischemia. More specifically, the amplitude of the T wave in the BXTY-0.5 g·kg
−1·d
-1, BXTY-2.0 g·kg
−1·d
-1, BXTY-8.0 g·kg
−1·d
-1, and BXTY-16.0 g·kg
−1·d
-1 groups, as well as in the nitroglycerin group, was significantly decreased compared with that in the model group (
Fig. 2(b)), while the heart rate of the BXTY-0.5 g·kg
−1·d
-1, BXTY-2.0 g·kg
−1·d
-1, BXTY-8.0 g·kg
−1·d
-1, BXTY-16.0 g·kg
−1·d
-1, and nitroglycerin groups was significantly increased compared with that of the model group (
Fig. 2(c)). Ischemia and hypoxia may cause myocardial damage and the release of myocardial enzymes into the blood, as well as significantly increasing the level of myocardial enzymes such as creatine kinase (CK) and lactate dehydrogenase (LDH) in the blood. At the same time, the free-radical damage caused by myocardial ischemia increases the membrane lipid peroxidation product malondialdehyde (MDA). As shown in
Figs. 2(d)-(f), the plasma levels of LDH, CK, and MDA increased significantly in the model mice, in comparison with those in the normal mice. The BXTY dose-dependently decreased the plasma levels of LDH and CK, and downregulated the MDA plasma concentration. These findings suggest that BXTY alleviates the symptoms of acute myocardial ischemia induced by pituitrin in mice.
3.3. BXTY dilates PE-pre-constricted mesenteric artery rings and suppresses PE-induced vasocontraction of mesenteric artery rings
Coronary artery spasm is a key cause of myocardial ischemia. Based on the clinical indication and components of BXTY, we predicted that BXTY might have effects on vasomotion, including vasodilation and vasoconstriction inhibition. Superior mesenteric arteries isolated from SD rats were used to check the effect of BXTY on vasomotion. Original trace showed that the PSS (12.5, 20.0, 50.0, or 100.0 μL) had no effects to the tension of endothelium-intact rat mesenteric arteries pre-contracted with PE (
Fig. 3(a)). Original trace showed that BXTY (2.5, 5.0, 10.0, or 20.0 μg·mL
−1) dilated vascular rings pretreated with PE in a dose-dependent manner to endothelium-intact rat mesenteric arteries pre-contracted with PE (
Fig. 3(b)). Original trace showed that BXTY (2.5, 5.0, 10.0, or 20.0 μg·mL
−1) dilated vascular rings pretreated with PE in a dose-dependent manner to endothelium-denuded rat mesenteric arteries pre-contracted with PE (
Fig. 3(c)). The statistical results confirmed that BXTY (2.5, 5.0, 10.0, or 20.0 μg·mL
−1) dilated vascular rings pretreated with PE in a dose-dependent manner, in both endothelium-intact (
Fig. 3(d)) and endothelium-denuded preparations (
Fig. 3(e)), in comparison with the treatment of the same volume of PSS. There was no statistical difference between the endothelium-intact and endothelium-denuded groups (
Fig. 3(e)). Similarly, BXTY pretreatment inhibited PE-induced vasoconstriction, in both endothelium-intact (
Figs. 3(f) and
(g)) and endothelium-denuded (
Figs. 3(h) and
(i)) superior mesenteric arterial preparations, in a dose-dependent manner. These findings suggest that the vessel-dilation effect of BXTY is independent of blood vessel endothelia, so subsequent experiments were carried out on rat endothelium-denuded superior mesenteric arteries.
3.4. BXTY dilates PE-pre-constricted mesenteric artery rings and suppresses PE-induced mesenteric artery ring contraction by activating sGC
sGC is a key enzyme in the nitric oxide (NO) signaling pathway. Activation of sGC increases the content of cGMP, a second messenger in MOVAs, activates cGMP-dependent protein kinases, and reduces intracellular Ca
2+ release and extracellular Ca
2+ influx, thereby relaxing vascular smooth muscle. As a target for the treatment of cardiovascular diseases, it is rapidly attracting interest and becoming a research hotspot in this field.
Fig. 4(a) is the original traces to show the vasorelaxation effect of BXTY (10.0 μg·mL
−1) to endothelium-denuded vascular rings pre-contracted with PE.
Fig. 4(b) is the original traces to show the inhibitory effect of NS 2028 (sGC inhibitor; 20 μmol·L
-1) pretreatment to BXTY-induced vasorelaxation in a PE-induced vasoconstriction model.
Fig. 4(c) is the original traces to show the inhibitory effect of ODQ (sGC inhibitor; 100 nmol·L
-1) pretreatment to BXTY-induced vasorelaxation in a PE-induced vasoconstriction model. The statistical results confirmed that BXTY (10.0 μg·mL
−1) dilated PE-pre-constricted endothelium-denuded artery rings (
Fig. 4(d)). Moreover, pre-incubation of endothelium-denuded vascular rings with the sGC inhibitors NS 2028 or ODQ partially eliminated the vasodilation effect of BXTY (
Fig. 4(d)).
Fig. 4(e) is the original traces to show the preventive effect of BXTY (10.0 μg·mL
−1) pretreatment for 10 min to PE-induced constriction of rat mesenteric arteries with denuded endothelium.
Fig. 4(f) is the original traces to show the inhibitory effect of NS 2028 (20 μmol·L
-1) pretreatment to the preventive effect of BXTY on a PE-induced vasoconstriction model.
Fig. 4(g) is the original traces to show the inhibitory effect of ODQ (100 nmol·L
-1) pretreatment to the preventive effect of BXTY on a PE-induced vasoconstriction model. The statistical results confirmed that BXTY (10.0 μg·mL
−1).
Fig. 4(h) is the original traces to show that pretreatment with 20 μmol·L
-1 NS 2028 had no effect on the PE-induced vasoconstriction of endothelium-denuded vascular rings.
Fig. 4(i) is the original traces to show that pretreatment with 100 nmol·L
-1 ODQ had no effect on the PE-induced vasoconstriction of endothelium-denuded vascular rings. The statistical results confirmed that BXTY pretreatment inhibited PE-induced vasoconstriction on endothelium-denuded vascular rings (
Fig. 4(j)), and NS 2028 and ODQ also greatly eliminated the inhibitory effect of BXTY on PE-induced vasoconstriction (
Fig. 4(j)). These findings suggest that BXTY dilates pre-constricted endothelium-denuded artery and inhibits the constriction of endothelium-denuded artery by activating sGC.
To further confirm the important role of sGC in the regulation of vasomotion by BXTY, MOVAs were incubated with BXTY for 3, 6, or 9 min; then, the protein expression of sGC was evaluated. Nitroglycerin was used as a positive control. Incubation with BXTY for 6 min resulted in the maximum increase in the protein level of sGC (
Fig. 4(k)). Therefore, 6 min of incubation time was selected for subsequent experiments. MOVAs were first treated with 20 μmol·L
-1 NS 2028 or 100 nmol·L
-1 ODQ for 24 h, followed by incubation with BXTY for 6 min. As shown in
Fig. 4(l), NS 2028 or ODQ pretreatment reversed the increase in sGC protein expression induced by BXTY. These results indicate that BXTY dilates pre-constricted artery and inhibits artery contraction by activating sGC.
3.5. BXTY suppresses vasomotion of mesenteric artery rings by increasing cGMP and PKG
cGMP is an important second messenger in MOVAs that can activate cGMP-dependent protein kinases, negatively regulate intracellular Ca
2+ release and extracellular Ca
2+ influx, and promote the dephosphorylation of myosin light chains, thereby relaxing vascular smooth muscle. Therefore, we investigated the role of cGMP in the regulation of vascular tension by BXTY. MOVAs were incubated with BXTY for 3, 6, or 9 min; then, the cGMP concentration in the supernatant was evaluated by means of an ELISA. Nitroglycerin was used as a positive control. The results showed that BXTY or nitroglycerin could increase the content of cGMP, and that BXTY time-dependently increased cGMP content in the cell supernatant (
Fig. 5(a)). MOVAs were first treated with an sGC inhibitor (20 μmol·L
-1 NS 2028 or 100 nmol·L
-1 ODQ) for 24 h, followed by incubation with BXTY for 9 min. As expected, NS 2028 or ODQ treatment inhibited BXTY-induced cGMP release (
Fig. 5(b)). PKG is a cGMP-dependent protein kinase in MOVAs that regulates vascular tension. To confirm the role of PKG in BXTY-mediated vasodilation, the selective PKG inhibitor KT 5823 was used to inhibit PKG activity in blood vessels and MOVAs. The results showed that the preincubation of endothelium-denuded vascular rings with 2 μmol·L
-1 KT 5823 for 5 min partially eliminated the vasodilation effect of BXTY on PE-mediated vasoconstriction (
Figs. 5(c)-(e)). Similarly, original trace showed that 2 μmol·L
-1 KT 5823 greatly decreased the inhibitory effect of BXTY on vasoconstriction induced by PE (
Figs. 5(f) and
(g)), while preincubating endothelium-denuded vascular rings with 2 μmol·L
-1 KT 5823 had no effect on vessel tension (
Fig. 5(h)). The statistical results confirmed the results (
Fig. 5(i)).
To further confirm the role of PKG in the regulation of vessel tension by BXTY, MOVAs were incubated with BXTY for 3, 6, or 9 min; then, the protein expression of PKG-1 was evaluated. Nitroglycerin was used as a positive control. Incubation with BXTY for 6 min resulted in the maximum increase in the protein level of PKG-1 (
Fig. 5(j)). In addition, MOVAs were first treated with 2 μmol·L
-1 KT 5823, 20 μmol·L
-1 NS 2028, or 100 nmol·L
-1 ODQ for 24 h, followed by incubation with BXTY for 6 min. As shown in
Fig. 5(k), KT 5823, NS 2028, or ODQ pretreatment reversed the increase in PKG-1 expression induced by BXTY. These results indicate that BXTY can dilate pre-constricted artery and inhibit artery constriction by activating the sGC-cGMP-PKG pathway.
4. Discussion
The results of this study confirm that BXTY can alleviate the symptoms of myocardial ischemia induced by pituitrin in mice, including ECG abnormalities and changes in plasma enzymes. BXTY promotes vasodilation and inhibits the abnormal vasoconstriction of the superior mesenteric artery in a dose-dependent manner, and this effect is independent of the function of blood vessel endothelia. In addition, pretreatment of the superior mesenteric artery with the sGC inhibitors NS 2028 or ODQ, or with the PKG inhibitor KT 5823, can reverse the vasodilatory effect of BXTY. Furthermore, BXTY increases the levels of sGC-β1 protein expression, cGMP, and PKG-1 expression in MOVAs, and NS 2028, ODQ, or KT 5823 can inhibit these effects of BXTY. These findings suggest that BXTY regulates vasodilation and contraction through the sGC-cGMP-PKG pathway in VSMCs, thereby improving myocardial ischemia.
The fundamental problem of myocardial ischemia is the imbalance between the coronary artery blood supply and myocardial oxygen demand. Coronary artery disorder is an important cause of myocardial ischemia [
12], including epicardial vasospasm/microvascular spasm, impaired coronary vasodilation, and enhanced vasoconstriction [
13]. Therefore, preventing vasospasm, avoiding abnormal vasoconstriction, and improving myocardial blood supply are effective strategies for treating myocardial ischemia.
Pituitrin was used to set up a myocardial ischemia mouse model in this research. Pituitrin is a neurohormone synthesized by neurons in the hypothalamus and is a drug widely used as a tool to induce myocardial ischemia by causing coronary artery spasm and contraction. The symptoms of myocardial ischemia induced by pituitrin in mice are similar to those caused by human coronary artery spasm in the clinic.
Most blood vessels consist of intima, media, and adventitia. VSMCs located in tunica media are an indispensable component of vascular structure [
14]. According to Poiseuille’s law, the lumen diameter regulated by vasomotor is the most important contributor to vascular resistance. Vasomotor depends on the contraction of VSMCs [
15]. Therefore, normal VSMC function plays an important role in vascular tension and blood transport. Biologically active fractions of BXTY, such as paeoniflorin [
16], salvianolic acid B [
17], salvianolic acid A [
18], and protocatechuic acid [
19], have been reported to produce vasodilative effects. We validated the effect of BXTY on vascular tension through a wire myograph system. PE is a sympathomimetic amine that acts predominantly on α-adrenergic receptors. It can increase systolic and diastolic blood pressure, as well as peripheral resistance. Therefore, PE was used to induce the acute vasoconstriction of superior mesenteric artery vessels, thereby simulating acute vasoconstriction during acute myocardial ischemia. The results showed that BXTY could relax the superior mesenteric artery contracted by PE in a dose-dependent manner. BXTY preincubation treatment could also antagonize the vasoconstrictive effect of PE in a dose-dependent manner. Moreover, BXTY preincubation treatment relaxed the superior mesenteric artery contracted by KCl in a dose-dependent manner (Fig. S1 in Appendix A). These findings indicate that BXTY alleviates acute myocardial ischemia caused by coronary vasospasm by inhibiting vasoconstriction and dilating acutely constricted blood vessels. In addition, the effects of BXTY on vessel tension are independent of vascular endothelia. Therefore, our subsequent mechanism research focused on VSMCs.
Vascular tension is regulated by the balance of vasodilators and vasoconstrictors [
20]. sGC is a key enzyme for vasodilation and a crucial target for the research and development of vasodilators. The heterodimeric sGC, which consists of α and β homologous subunits, is a main physiological senser expressed in VSMCs, endothelial cells, and cardiac myocytes [
21]. In the vascular system, sGC plays a major role in regulating vascular tension through endogenous and exogenous signals. It assists in communication between VSMCs and endothelial cells through NO [
22]. The NO produced by the endothelium and enzymatic activity modulators regulates the activity of sGC, which converts the cyclization of 5′-guanosine triphosphate (GTP) into second messenger cGMP in VSMCs by changing the conformation and activating the catalytic site of sGC [
23], [
24]. cGMP then activates PKG, which produces vasodilation by inhibiting extracellular Ca
2+ influx, stimulating Ca
2+ uptake through sarcoplasmic reticulum Ca
2+ adenosine triphosphatases (ATPase) and reducing the Ca
2+ sensitivity of myofilaments [
25], [
26].
Aside from being regulated by NO, sGC stimulators and sGC activators can also directly bind to sGC to enhance NO signaling [
27]. sGC stimulators enhance the enzymatic activity of sGC in a NO-independent manner and have the capacity to synergize with endogenous NO. sGC activators bind to heme-free or oxidized sGC [
28]. Dysfunction of NO-sGC cascade signaling leads to numerous pathological processes, such as cardiovascular disease, hypertension, asthma, and neurodegeneration [
29]. Targeting sGC for the treatment of cardiovascular disease has become a focus of research and the development of novel therapeutic agents. Riociguat, a first-in-class sGC stimulator, has been approved for the treatment of patients with pulmonary arterial hypertension and chronic thromboembolic pulmonary hypertension [
30], [
31]. Vericiguat is another sGC stimulator that is widely used in the treatment of heart failure; it increases the level of cGMP by directly acting on sGC and improves the sensitivity of sGC to NO by stabilizing their binding site [
32], [
33]. The sGC activator cinaciguat is designed for the treatment of heart failure. Ataciguat (HMR1766), which is used to improve blood pressure and orthostatic tolerance, is in the pre-approval stage, as are several other chemicals targeting sGC [
34]. Thus, targeting sGC is a promising strategy for the treatment of ischemic cardiovascular disease.
Calcium-activated potassium channels (K
Ca), ATP-sensitive potassium channels (K
ATP), L-type calcium channels (LTCCs), Ca
2+-ATPase (SERCA), and inositol 1,4,5-trisphosphate receptors (IP
3R) also play important roles in the regulation of vascular tension [
35]. We excluded their roles in the BXTY regulation of vascular tension through experiments. Pretreatment with 12 mmol·L
-1 diltiazem, 5 μmol·L
-1 A23187, 100 μmol·L
-1, 2-aminoethyl diphenylborinate (2-APB), and 10 μmol·L
-1 thapsigargin, respectively, did not affect the vasodilative effect of BXTY on endothelium-denuded vascular rings, which excluded the roles of LTCCs, Ca
2+ release, IP
3R, and SERCA (Fig. S2 in Appendix A). Furthermore, pretreatment with 10 μmol·L
-1 glibenclamide or 100 μmol·L
-1 tetraethylammonium chloride (TEA), respectively, did not affect the vasodilative effect of BXTY on endothelium-denuded vascular rings, which excluded the contributions of K
ATP and K
Ca (Fig. S3 in Appendix A).
However, preincubation with the sGC inhibitors NS 2028 or ODQ, or with the PKG inhibitor KT 5823, obviously reduced the vasodilative effect and the inhibitory effect of BXTY on the vasoconstriction of vascular rings, suggesting that the anti-vasoconstrictive effect of BXTY is mediated by the sGC-cGMP-PKG signaling pathway. This conclusion was further supported by the finding that BXTY elevated the levels of sGC-β1, cGMP, and PKG-1 in the lysates of MOVAs, which was reversed by pretreating MOVAs with NS 2028, ODQ, or KT 5823.
5. Conclusions
This study investigated the pharmacological effects of BXTY in the treatment of myocardial ischemia in a mouse model. Our findings demonstrate that BXTY can alleviate the symptoms of myocardial ischemia. The effect of the preventive administration of BXTY is equivalent to that of the therapeutic administration of nitroglycerin—the classic clinical anti-myocardial ischemia drug. Inhibiting abnormal vasomotor is one of the underlying mechanisms of the cardioprotective effects of BXTY. Activating sGC, increasing intracellular cGMP content, and increasing the activity of cGMP-dependent PKG in vascular smooth muscle is the molecular signaling mechanism for BXTY (
Fig. 6). We used modern pharmacological techniques to clarify the pharmacological effects and action mechanisms of traditional drugs, thereby facilitating the communication between traditional medicine and modern medical systems and promoting drug research and development.
Acknowledgments
This work was supported by the National Natural Science Foundation of China (81870259, 82170431, 81903608, and U21A20339), the CAMS Innovation Fund for Medical Sciences (CIFMS; 2019-I2M-5-078), and the Postdoctoral Research Foundation of Heilongjiang Province (LBH-Q20148).
Compliance with ethics guidelines
Shuang Yang, Yixiu Zhao, Xiaoling Cheng, Tingting Zhan, Jiaying Tian, Xue Liu, Chunyue Ma, Zhiqi Wang, Luying Jin, Qian Liu, Yanli Wang, Jian Huang, Jinhui Wang, Yan Zhang, and Baofeng Yang declare that they have no conflict of interest or financial conflicts to disclose.
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.eng.2023.06.009.
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