STDPs were supplied by the Inner Mongolia Conba Pharmaceutical Co., Ltd. (Shanghai, China) and approved for marketing in 2008, with approval number: Z20080018. STDPs consist of seven active pharmaceutical ingredients, which was first dissolved in dimethyl sulfoxide (DMSO) with an initial concentration of 42 mg/ml (STDP:DMSO = 42 mg:1 ml). The resulting solution was autoclaved and stored at −20°C until further use. The fingerprint was established by HPLC to control the quality of STDPs ( Supplementary Figure S1 ). LY294002 (154447-36-6, Selleck.cn, USA), the inhibitor of PI3K, was purchased from Selleck.co.uk. Telmisartan was indicated to be effective for patients with myocardial ischemia and used as a positive drug (J20090089, Boehringer Ingelheim, Germany). LPS (l2880, biodee, China) was purchased from the Beijing BioDee Biotechnology Co., Ltd.

Experimental procedures were conducted in accordance with the China Physiological Society’s “Guiding Principles in the Care and Use of Animals,” and approved by the animal care committee of the Beijing University of Chinese Medicine (BUCM-4-2021113002-4048). Fifty Sprague–Dawley (SD) rats (220–240 g) were purchased from the Spelford (Beijing) Laboratory Animal Co., Ltd. All rats were on adaptive feeding for 3 days and fasted for 12 h before the surgery. The left anterior descending (LAD) artery of the rats were ligated to induce acute myocardial ischemia and CMD model as previously described (Wang et al., 2013). The rats were randomly divided into the sham group, model group, STDP group (21.6 mg/kg*day), and telmisartan group (8.23 mg/kg*day) for 7 days. An equal amount of saline was given by gavage in the other groups. Pentobarbital sodium was used as an anesthetic during the surgeries, and the greatest effort was made to minimize pain.

Rats were anesthetized and fixed on a wooden board. Cardiac function was assessed using an echocardiograph (Vevo TM 2100, Visual Sonics, Canada). The main parameters included the left ventricular ejection fraction (LVEF), short-axis shortening fraction (LVFS), left ventricular end-diastolic internal diameter (LVID; d), and left ventricular end-systolic internal diameter (LVID; s).

Myocardial tissue from each group was fixed in 4% paraformaldehyde, and paraffin-embedded sections were prepared. Sections were stained with hematoxylin–eosin (HE) as previously described (Meng et al., 2021).

In this study, OMAG imaging system (BaslerSPL 4096-140 km, Germany) based on optical coherent tomography (OCT) technology was used to evaluate the density of microvessels in the rat heart (Wan et al., 2016). Briefly, the spectral bandwidth was 100 nm, the central wavelength was 1,310 nm, and the axial resolution was 15 μm. The OCT system used an interframe ultra-high sensitivity OMAG algorithm to extract 3D vessels from images of cardiac tissue structures. The maximum intensity projection (MIP) of the volumetric vasculature allowed the morphological information of the microvascular network to be displayed from the top view (Redd et al., 2019). Each data volume took 1 min. The rat was fixed to a wooden board after anesthetization, and a small animal ventilator was attached to ensure that the rat could breathe normally. The heart was exposed by an opening between the third and fourth intercostal space. An appropriately sized cotton pad moistened with saline was placed under the heart to raise the heart slightly for the acquisition of microvascular imaging. The probe was placed in the same position in the heart, and the heart was held at pressures for image acquisition.

Paraffin sections of heart sections were dewaxed and blocked with serum for 30 min. The sections were incubated overnight at 4°C with anti-CD206 antibody (ab201340, Abcam, USA) and anti-CD31 antibody (ab53004, Abcam, USA) in a wet box. Then the slides were washed with PBS three times. Secondary antibody (D001-34, Abcam, USA) was incubated for 50 min at room temperature in the dark. The nuclei were stained with DAPI (C0065, Solarbio, China).

Cells were seeded in confocal dishes, fixed with 4% paraformaldehyde for 15 min, and permeabilized with 0.5% TritonX-100 for 20 min. The cells were blocked with 1% BSA in a 37°C incubator for 1 h. Incubation with anti-CD206 antibody at 4°C overnight was followed by incubation with the anti-rabbit fluorescent secondary antibody IgG (AB0141, Abways, China) for 1 h at room temperature. Nuclei were stained with DAPI for 5 min.

Rat heart tissues and cell lysates were prepared using RIPA lysis buffer containing protease inhibitor (c1053-100, Applygen, China), and phosphatase inhibitors (p1260-1, Applygen, China). An equal amount of protein was separated on 10%–15% SDS-polyacrylamide gels and transferred onto PVDF membranes. After blocking with 5% nonfat milk at room temperature for 2 h, the PVDF membranes were incubated with the appropriate primary antibody, followed by a secondary antibody. The PVDF membrane was exposed to ECL reagents (p1020-25, Applygen, China) to visualize the protein signals. The antibodies used for Western blot were as follows: anti-PI3K (1F6A7, Proteintech, USA), anti-Akt (2C5D1, Proteintech, USA), anti-Phospho–PI3K (CY6427, Abways, China), anti-Phospho–Akt (P31749, Abways, China), anti-VEGFA (ab 214424, Abcam, USA), anti-ARG-1 (P05089, Abways, China), anti-mTORC1 (Q6UUV9, Abways, China), anti-CD206 (JF0953, HuaAn, China), anti-rabbit IgG H and L (ab0101, Abways, China), and anti-mouse IgG H and L (ab0102, Abways, China).

The plasma levels of troponin (cTnI), IL-10, and VEGF-A were quantified using the ELISA kits (Wuhan Yunclone Technology Co., Ltd., Wuhan, China) according to the manufacturer’s instructions.

HUVECs were obtained from the Beijing Anzhen Hospital. HUVECs were cultured with a 1640 medium containing 10% FBS and 1% penicillin/streptomycin (P/S) in a humidified incubator with 5% CO₂ at 37°C. The groups were as follows: control group, model group, STDP group (100 ng/ml), and VEGF-A group (20 ng/ml). In the model group, oxygen–glucose deprivation–reperfusion (OGD/R) was constructed to induce cell injury. HUVECs were seeded at a density of 8 × 10³/well in 96-well plates. After 24 h, cells in the model group were incubated with Earle’s balanced salt solution in an anaerobic box for 6 h. Then the Earle’s balanced salt solution was removed, and 1640 medium was added to recover oxygen–glucose for 12 h at 37°C with 5% CO₂. The cells in the STDP and VEGF-A groups underwent ODG/R simultaneously with STDPs or VEGF-A treatment.

Cell viability was measured using CCK8 (Jiancheng, Nanjing, China) according to the manufacturer’s instructions. After different treatments, 10 µl of CCK8 was added to the well, and the plate was incubated in 37°C for 2 h. Then absorbance was measured using a microplate reader (PerkinElmer VICTOR1420, USA) at 450 nm.

When HUVECs were cultured to 80%–90% confluence in a six-well plate, a line was gently drawn on the bottom of each well with a pipette and washed three times with PBS. The width of the scratch was measured after different treatments. The degree of the scratch healing was observed under an inverted microscope (BX50-FLA, Olympus, Tokyo, Japan).

The 96-well plates were coated with 35 μl of growth factor-reduced Matrigel (354230, Biocoat, USA). After different treatments, HUVECs were seeded at 1 × 10⁶ cells/ml onto the Matrigel. After 5 h, the supernatant was discarded, and the cells were washed with PBS. Calcein-AM dye (5 μg/ml) was added and stained for 5 min. Photographs were taken using an inverted fluorescence microscope (SP8, Leica Microsystems CMS GmbH, Germany).

The 10-day-old SD rats were sterilized in beakers containing 75% ethanol for 3–5 min. The tibia and femur of the rats were stripped, and the bone marrow was squeezed out with forceps into the centrifuge tube with 1 ml of MEM (10% FBS, 1% P/S, M-CSF 10 μg/ml). Fivefold the amount of erythrocyte lysis solution was added, and the tube was placed at 4°C for 5 min, then centrifuged at 1,000 rpm/min for 3 min. The supernatant was discarded, the cells were resuspended by complete MEM medium and inoculated in 10-cm² culture dishes. After 1 h, the supernatant was collected and inoculated in the corresponding culture plates, and half of the liquid volume was renewed after 2 days.

The groupings were as follows: control group, model group, STDP group, LY29 group, and LY29 + STDP group. BMDMs in the model group were treated with LPS (Beijing Biodee Biotechnology, 10 μg/ml) for 24 h. BMDMs in the inhibitor group were treated with LY294002 (154447-36-6, Selleck, USA) at a dose of 10 μM for 24 h. STDPs were administered at a dose of 400 µg/ml.

BMDMs were cultured to 80%–90% confluence in a 96-well plate. After different treatments, the cell supernatant was extracted and assayed according to the NO assay kit method (bn27106-500, Biorigin, China). After adding the standards/samples, Griess reagent I and Griess reagent II, the plate was mixed well and incubated at room temperature for 5 min. NO concentration was measured at 540 nm using a microplate reader.

BMDMs were enzymatically digested for 40 s and prepared into a single-cell suspension. After centrifugation, the supernatant was discarded, and the cells were washed with PBS and centrifuged again. Anti-CD206 antibody (sc58986 PE, Santa Cruz, CA, USA) was added, and the BMDMs were incubated on ice for 40 min. After being washed twice, BMDMs were suspended in 300 μl of PBS and then detected by flow cytometry (Canton II BD, USA).

All data were expressed as mean ± SD. Statistical analysis was performed using GraphPad Prism 6. Statistical analysis was performed using one-way analysis of variance (ANOVA). Dunnett’s test was used for multiple comparisons between groups. Values of p < 0.05 were considered statistically significant.

To determine whether STDPs could attenuate cardiac dysfunction in CMD rats induced by LAD ligation, echocardiographic assessment, H and E staining, and ELISA assay were performed. The experimental protocol is presented in Figure 1A. Rats in the model group showed a significant reduction in cardiac function (Figures 1B,C). As shown by the echocardiography results, EF was decreased by 57.05%, and FS was decreased by 67.26% in the model group compared with the sham group, which were remarkably reversed by STDP administration. H&E staining of heart sections showed an obvious increase in vascular tube-like formation in the STDP group (Figure 1D). In addition, serum cTnI, an indicator of myocardial injury, was detected, and STDPs could diminish the release of cTnI into the serum compared with the model group (Figure 1E). Telmisartan was used as a positive drug. These results suggested that STDPs improved cardiac function in LAD ligation-induced CMD rats.

The real-time perfusion of cardiac vessels in the different groups was assessed by OMAG, and the representative images are presented in Figure 2A. After STDP treatment, microvessel density increased significantly compared with the model group (Figure 2B). It is well known that M2 macrophages participate in the process of heart repair and angiogenesis (Hong and Tian, 2020), which are marked by CD206 and characterized by releasing high levels of Arg-1 and IL-10 (Wu et al., 2019). Therefore, the effects of STDPs on the polarization of M2 macrophage were explored. IF staining showed that compared with the model group, the number of M2 macrophages in the STDP group was upregulated ( Supplementary Figure S2 ). Meanwhile, STDPs increased the expression of Arg-1 and CD206 in ischemic myocardial tissue compared with the model group (Figure 2D). STDP also could stimulate the release of IL-10 into the serum (Figure 2E). Interestingly, we found that increased M2 macrophages were around endothelial cells (Figure 2C), accompanied with an increased VEGF-A expression in the STDP group compared with the model group (Figure 2D). These results indicated that STDPs could promote angiogenesis by inducing M2 macrophage polarization in ischemic myocardial tissues.

Phosphorylation-activated PI3K/Akt was critically involved in macrophage polarization in heart tissues (Vergadi et al., 2017); thus, the effects of STDP on the expressions of p-PI3K and p-Akt were examined. Results showed that the expressions of p-PI3K and p-Akt were significantly decreased in the model group compared with the sham group. After treatment with STDPs, levels of p-PI3K and p-Akt were upregulated ( Figures 3A,B ). Meanwhile, treatment of STDP could reverse the low expression of mTORC1 in the model group (Figure 3C). These data indicated that STDP could induce M2 macrophage polarization via regulating the PI3K/Akt/mTORC1 pathway.

In order to explore the effect of STDPs on angiogenic properties of endothelial cells in vitro, we established the ODG/R-induced HUVECs model. The treatment of HUVECs with STDPs (10–200 ng/ml) for 24 h significantly enhanced cell viability compared with the model group ( Figure 4A ). Among them, 100 μg/ml of STDPs was the best concentration for subsequent experiments to evaluate the effects and mechanism (Figure 4B ). The proliferation and migration of HUVECs in the model group were reduced, which were upregulated after STDP treatment (Figures 4C,D). The effect of STDPs on the angiogenetic properties of endothelial cells was further examined using the tube formation assay. The tube length and numbers of junctions treated with STDPs were significantly increased compared with the model group, and VEGF-A was used the positive control ( Figure 4E ). These data suggested that STDPs could promote the angiogenesis of HUVECs directly.

To further clarify the effects of STDPs on macrophage polarization-induced angiogenesis, LPS-induced BMDM inflammation model was used. CCK8 result showed that STDPs (10, 100, and 1,000 μg/ml) had no cytotoxicity on BMDMs ( Figure 5A ). The effective concentrations of STDPs were assessed by the NO level in the cell supernatant. STDPs (400 μg/ml) had the best inhibitory effect on the release of NO after LPS stimulation (Figure 5B) and were used in the subsequent experiments. IF staining showed that STDPs obviously increased CD206 expression in LPS-induced BMDMs (Figure 6A). Similarly, CD206-postive BMDMs detected by flow cytometry were upregulated after STDP treatment, which were identical to the IF results (Figure 6B). The expression of Arg-1 in the STDP group was increased compared with the model group (Figure 6C). In addition, STDPs enhanced the expression of VEGF-A (Figure 6D) and the release of VEGF-A in the supernatant (Figure 6E). These results revealed that STDPs modulated M2 macrophage polarization and increased VEGF-A release, which played important roles in angiogenesis.

Furthermore, the effective mechanism of STDPs on macrophage polarization-induced angiogenesis was assessed. Immunoblot results showed that STDPs could promote the expressions of p-PI3K and p-Akt in LPS-induced BMDMs compared with the model group (Figures 5D, E). In contrast, the expression of mTORC1 was increased in the model group, and STDP treatment inhibited mTORC1 expression (Figure 5F). Then LY294002, the inhibitor of PI3K, was applied. LY294002 reversed the effects of STDPs on NO release, and the expressions of p-PI3K, p-Akt, and mTORC1 (Figures 5C–F). At the same time, the enhancement of STDPs on M2 macrophage polarization and VEGF-A release could both be reversed by LY294002 (Figure 6E). Therefore, STDPs modulated M2 macrophage polarization and increased VEGF-A release via the PI3K/AKT/mTORC1 pathway in LPS-induced BMDMs ( Figure 7 ).