Abstract
Background
Vascular injury is a major contributor to the development of cardiovascular diseases. Following vascular damage, macrophages migrate to the injury site and, during the later stages of vascular repair, secrete cytokines such as interleukin-10 (IL-10) and transforming growth factor-β1a (TGFB1A), thereby promoting vascular regeneration. Previous studies have demonstrated that macrophage recruitment to sites of tissue injury is mediated by the CXCR4A-CXCL12B signaling axis. In a screening of traditional Chinese medicinal herbs for cardiovascular therapeutic potential, Salvia miltiorrhiza root was identified as a promising source of bioactive compounds capable of enhancing vascular repair through modulation of the CXCR4A-CXCL12B axis.
Methods
Establishing a vascular injury model in transgenic zebrafish lines Tg (flk1:eGFP; gata1:dsRed) using a two-photon microscopy laser system. Dynamic monitoring of vascular repair via two-photon microscopy. Evaluate macrophage migration capacity in a Tg (mpeg1:eGFP) zebrafish vascular injury model using confocal microscopy. Detection of il-10 and tgfb1a expression released by macrophages via qPCR experiments. Detect CXCR4A-CXCL12B expression at the site of zebrafish vascular injury via fluorescence in situ hybridization coupled with antibody staining.
Results
We confirm that compounds from the selected extract promote macrophage migration to vascular injury sites by upregulating the CXCR4A-CXCR12B signaling axis. This process accelerates repair of damaged blood vessels in zebrafish by inducing the release of cytokines such as il-10 and tgfb1a.
Conclusions
This study confirms that Salvia miltiorrhiza, a traditional Chinese medicinal plant, is a valuable source of bioactive compounds with pro-angiogenic properties. Our findings provide scientific support for the traditional use of Salvia miltiorrhiza active components in treating vascular injuries.
Keywords: CXCR4A-CXCL12B, macrophages, tanshinone IIA, vascular repair, zebrafish
1. Introduction
Cardiovascular diseases (CVDs) occur with increasing frequency worldwide and remain a leading cause of mortality. Vascular dysfunction plays a central role in the development of many CVDs, making the restoration of vascular health a critical therapeutic goal (1). Endothelial cells (ECs) are key regulators of vascular remodeling, maintaining homeostasis by mediating vasodilation, contraction, and overall vascular function (2, 3).
Current pharmacological treatments for CVD include β-blockers, calcium channel blockers, angiotensin-converting enzyme inhibitors, anticoagulants, diuretics, and platelet aggregation inhibitors (4). Although effective, these agents are often limited by serious adverse effects. For example, high-dose β-blockers have been associated with harmful outcomes in high-risk myocardial infarction patients with heart failure or cardiogenic shock (5), and statins can cause complications such as rhabdomyolysis (6). Consequently, traditional Chinese medicine has attracted growing attention as a complementary or alternative therapy, owing to its long history of clinical use and generally fewer side effects (7).
Salvia miltiorrhiza Bunge (The plant name originates from http://mpns.kew.org November 10, 2025), a perennial herb of the Labiatae family (8), exhibits broad biological activities, including antibacterial, antioxidant, and anti-inflammatory effects. It is widely used to treat hyperlipidemia, stroke, and both cardiovascular and cerebrovascular diseases (9). Among its active constituents, Tanshinone IIA (TAN IIA)—a fat-soluble diterpene—shows particularly potent cardiovascular and cerebrovascular protective effects, including inhibition of atherosclerosis (AS) (10). TAN IIA reduces oxidative stress and inflammatory damage in ECs improves endothelial dysfunction, and inhibits proliferation and migration of vascular smooth-muscle cells (VSMCs), thereby limiting vascular stenosis (11). It has also been reported to attenuate atherosclerosis through macrophage-mediated pathways (12). Despite these findings, the pharmacological mechanisms of TAN IIA remain incompletely understood, and its role in vascular repair is largely unexplored.
Macrophages are essential mediators of vascular regeneration. Their remarkable plasticity allows them to adapt to diverse microenvironments and adopt distinct functional phenotypes. In response to specific stimuli, macrophages polarize toward either the pro-inflammatory M1 or the anti-inflammatory, pro-repair M2 phenotype through complex signaling and transcriptional networks. Macrophages coordinate angiogenesis, inflammation, and extracellular matrix remodeling during all stages of tissue repair, from the initial inflammatory response to resolution and remodeling (13–16). The transition from M1 to M2 macrophages is particularly important for wound healing and tissue regeneration (17). M2 macrophages, induced by cytokines such as IL-4 and IL-10 (18–21), secrete high levels of anti-inflammatory mediators including IL-10 and TGF-β, thereby suppressing inflammation and promoting repair (22–29). They also facilitate extracellular matrix synthesis and remodeling—producing fibronectin, collagen, and proteoglycans—to support tissue regeneration (30–32). In addition, M2 macrophages efficiently clear apoptotic cells and debris, helping to resolve inflammation and restore tissue homeostasis (33–36). At later stages, they regulate metabolic processes, promote angiogenesis, and further remodel the extracellular matrix (37–42). Remarkably, M2 macrophages can even differentiate into endothelial-like cells and contribute directly to neovascularization, a process mediated by transcription factors such as Prox1 (43, 44). Consistent with these roles, macrophages have been shown to mediate vascular repair in zebrafish models (45).
The present study aimed to establish a zebrafish vascular-injury model to evaluate the effects of TAN IIA on vascular repair. Our results demonstrate that TAN IIA significantly accelerates the healing of injured vessels. Mechanistically, TAN IIA enhances macrophage recruitment to injury sites and promotes their conversion to M2 macrophages via the CXCR4A-CXCR12B signaling axis, thereby facilitating vascular repair in zebrafish.
2. Materials and methods
2.1. Zebrafish
Use transgenic zebrafish lines Tg (flk1:eGFP; gata1:dsRed), Tg (flk1:eGFP), and Tg (mpeg1:eGFP) at 72 hpf (hours post-fertilization). These lines were bred in-house. The transgenic zebrafish strains Tg (flk1: eGFP; gata1: dsRed), Tg (flk1: eGFP), and Tg (mpeg1: eGFP) were maintained and reared under standard laboratory conditions at 26 ± 2 °C. The transgenic adult zebrafish and zebrafish embryos used in this experiment were randomly selected. The study complies with the Animals management regulations (Order No. 2 of the State Science and Technology Commission of the People’s Republic of China, 1988). Permit No. 2403704.
2.2. Main reagents
Drug tanshinone IIA (Glpbio, USA, purity >99.50%), carboxymethyl cellulose sodium (Coolaber, China), DMEM basal medium (Gibco, USA), AMD3100 (MCE, USA), SDF-1β (MCE, USA). JAK1 Protein Antibody (UpingBio, CHINA), p-JAK1 Protein Antibody (Servicebio, CHINA), STAT6 Protein Antibody (UpingBio, CHINA), p-STAT6 Protein Antibody (UpingBio, CHINA).
2.3. Establishment of a zebrafish vascular injury model
Establish a venous vascular injury model in transgenic zebrafish lines Tg (flk1: eGFP; gata1: dsRed), Tg (flk1: eGFP), and Tg (mpeg1: eGFP) by a two-photon laser scanning fluorescence microscopy system. First, zebrafish embryos at 72 hpf were fixed with carboxymethyl cellulose sodium in a Cell Culture Dish (NEST, China) and placed on a microscope stage. A two-photon laser scanning fluorescence system was activated to observe the expression of Tg (flk1:eGFP; gata1:dsRed), Tg (flk1:eGFP), and Tg (mpeg1:eGFP). Select the tail vein terminal portion of the zebrafish (length 110 μm) as the laser focus site and use a 920 nm infrared laser to irradiate the focus site for 2 seconds to establish a transgenic zebrafish vein injury model.
2.4. Fluorescence in situ hybridization coupled antibody staining
Detection of cxcl12b and cxcr4a gene expression in damaged blood vessels through fluorescence in situ hybridization coupled antibody staining (46).
2.5. Cell culture and preparation of conditioned medium
Cells from the human endothelial cell line EA. hy926 and the mouse macrophage cell line RAW264.7 were cultured in DMEM basal medium supplemented with 10% fetal bovine serum (FBS), antibiotics (100 mg/mL streptomycin and 100 U/mL penicillin). Both cell lines were cultured at 37 °C in a humidified incubator containing 5% CO2. EA. hy926 cells were seeded into 6-well plates (1.5 × 104 cells/well). When the cell density reached 80%, the cells were treated with 1 μg/mL LPS (Sigma, USA) for 24 hours, subsequently treated with TAN IIA (0.5, 1, and 2 μM), and finally cultured in DMEM basal medium supplemented with 0.5% FBS for 24 hours. Then, collect the CM from each group and centrifuge at 2000×g for 10 minutes to collect the supernatant, which is stored at -80 °C for further experiments.
2.6. Transwell migration assay
A total of 200 μL of RAW264.7 cell suspension (containing 0.5% FBS DMEM medium) was seeded into the upper chamber of an 8 μm pore size transwell chamber (Corning, USA) at a density of 1 × 105 cells/chamber. The cells were co-cultured with 600 μL of conditioned medium (CM), AMD3100 (10 μM) and SDF-1β (10 μM) from different groups in the lower chamber for 24 hours. Residual macrophages that did not migrate into the upper chamber were removed using a sterile cotton swab. Migrated cells were fixed with 4% PFA for 20 minutes and stained with 0.1% crystal violet (Solarbio, China) for 15 minutes. Finally, migrated macrophages were counted using ImageJ software.
2.7. Wound healing
RAW264.7 cells (4 × 105 cells/well) were seeded in a 6-well culture plate (Corning-Costar, USA) for 24 hours to reach 80% confluence, followed by serum starvation for 12 hours. A wound line was created using a 200 μL micropipette tip. CM, AMD3100 (10 μM) and SDF-1β (10 μM) were then added to the 6-well plate, and cells were cultured for 24 h. The wound area was visualized under a microscope and quantified using ImageJ software.
2.8. Molecular docking
Obtain cxcl12b (Q6V9B5), cxcr4a (Q7ZU02) and IL 4 (D1YSM1) from the RCSB Protein Data Bank (https://www.rcsb.org) and download AlphaFold (https://alphafold.ebi.ac.uk/). The experimental ligand TAN IIA was sourced from the PubChem database (https://pubchem.ncbi.nlm.nih.gov/) and converted to pdbqt format using OpenBabel software. After processing the molecular structures using PyMOL 3.1.6 and AutoDockTools 1.5.6, the most favorable free energy binding conformation was selected based on the principles described in AutoDock Vina (http://vina.scripps.edu). Visualization was performed using Discovery Studio 2025 Client.
2.9. Network pharmacology analysis
Retrieve known targets and SMILES molecular structures for TAN IIA from the DGIdb (https://dgidb.org/), TCMSP (https://www.tcmsp-e.com/), and PubChem (https://pubchem.ncbi.nlm.nih.gov/) databases. Subsequently, the SMILES number was imported into the SwissTargetPrediction (http://www.swisstargetprediction.ch/) online platform to screen for potential targets based on a probability threshold greater than zero. Finally, all target sources were integrated and deduplicated, yielding a total of 100 potential targets for TAN IIA. Identified 3,737 unique targets associated with vascular injury from the Genecards (www.genecards.org), MalaCards (https://www.malacards.org/), and OMIM (https://www.omim.org/) databases. The 100 potential targets for TAN IIA identified in the study were subjected to an intersection analysis with 3,737 targets associated with vascular injury. By plotting a Venn diagram, a total of 60 core targets co-present in both datasets were identified. Import the above intersecting targets into the STRING database (https://cn.string-db.org/), set the confidence threshold to 0.4, and exclude free-floating targets to obtain the protein interaction network.
2.10. Quantitative real-time polymerase chain reaction analysis
Total RNA was extracted from transgenic zebrafish and EA. hy926 cells using TRIzol (Thermo, USA), and cDNA was obtained from 1000 ng of total RNA using a reverse transcription kit (Thermo, USA). Then, q-PCR was performed using SYBR Green qPCR pre-mix (Thermo, USA). Relative gene expression was calculated using the 2-ΔΔCT method, with β-actin as the standardized reference. The primers used in this study are listed in Supplementary Table 1.
2.11. Western blot
After laser-damaging the blood vessels of 3dpf Tg (flk1:eGFP;gata1:dsRed) larvae, proteins were extracted from whole fish using a protein extraction kit (NE-PER™ Nucleus and Cytoplasmic Extraction Reagent, Thermo Scientific). The extracted proteins were subjected to SDS-PAGE electrophoresis following concentration measurement, then transferred to a PVDF membrane.
2.12. Statistics
All data are expressed as mean ± standard error of the mean (SEM). Statistical analysis of two groups of samples was performed using Student’s t-test with GraphPad Prism 9 software (GraphPad Software, USA). Statistical analysis of more than two groups of samples was performed using one-way analysis of variance (ANOVA). In all cases, P < 0.05 was considered statistically significant.
3. Results
3.1. Construction of a zebrafish venous vessel injury model
To establish a venous vascular injury model, zebrafish embryos (72 hpf) from the transgenic line Tg (flk1:eGFP;gata1:dsRed) were immobilized in culture dishes with carboxymethyl cellulose. Vessel structures were visualized using two-photon laser scanning fluorescence microscopy. Target vessels were identified and marked, then injured with the microscope’s infrared laser system (Figure 1A). Successful vessel damage was confirmed a localized loss of eGFP fluorescence (Figure 1B; Supplementary Video 1) and by visible surface lesions under bright-field microscopy. These observations confirmed the successful establishment of a zebrafish venous vessel injury model.
Figure 1.
Zebrafish vascular injury model. (A) Schematic diagram of establishing a venous vascular injury model using zebrafish embryos (72 hpf). (B) The vascular structure and red blood cells can be clearly observed under a two-photon microscope. The tail vein returning to the heart of the zebrafish is marked with an infrared laser system, and the marked vessels are damaged at specific points using infrared laser to construct a zebrafish vascular damage model. Green marks the vessels, red marks the red blood cells, and red ellipses mark the vessels that need to be damaged. The blue arrow indicates the area of vascular damage under white light.
3.2. TAN IIA promotes vascular repair
Using this model, we examined the effects of TAN IIA—the major bioactive component of Salvia miltiorrhiza Bunge—on vascular repair. Injured zebrafish were treated with TAN IIA, and vascular recovery was assessed at 2, 4, and 6 hours post-injury, with DMSO serving as vehicle control. By 4 hours, TAN IIA (2 μM)-treated fish exhibited markedly faster vascular regeneration than controls (47); by 6 hours, damaged vessels were largely restored, whereas DMSO-treated vessels remained incompletely repaired (Figures 2A, B). q-PCR analysis revealed that vascular remodeling-related gene (mmp9, vegfaa, pdgfba, arg1, angpt1) were significantly upregulated in the TAN IIA treatment for 6 hours after vascular injury in zebrafish (Figures 2C, D). Collectively, these results indicate that TAN IIA accelerates the vascular repair process in zebrafish.
Figure 2.
TAN IIA promotes rapid repair of damaged blood vessels in zebrafish. (A, B)TAN IIA accelerates the repair of damaged blood vessels in zebrafish. The white triangular arrows indicate the repaired blood vessels. (C, D) The effect of TAN IIA (2 μM) and dmso (2 μM) on the mRNA expression levels of mmp9, vegfaa, arg1, pdgfba, and angpt1 genes. DMSO group refers to zebrafish that did not undergo blood vessels damage. * P < 0.1 versus control group, ** P < 0.01 versus control group, *** P < 0.001 versus control group, **** P < 0.0001 versus control group.
3.3. TAN IIA enhances macrophage recruitment in zebrafish
To explain the mechanism by which TAN IIA promotes rapid repair of damaged blood vessels, we focused on macrophages. Previous studies have shown that TAN IIA can slow the progression of cardiovascular disease by modulating macrophage activity, and macrophages, particularly M2 macrophages, are known to participate in vascular and tissue repair (17). To determine whether TAN IIA acts on macrophages during vascular repair, we employed Tg (mpeg1: eGFP) transgenic zebrafish subjected to vascular injury (Figure 1). At 6 hours, TAN IIA-treated larvae displayed a significantly greater number of macrophages recruited to the injury site compared with DMSO-treated controls (Figures 3A, B). Moreover, qPCR analysis revealed that TAN IIA treatment for 6 hours significantly up-regulated multiple macrophage and M2 related genes, including general macrophage markers (mpeg1 and mfap4), the M2 surface markers (mrc1a), the polarization factor (il-4), anti-inflammatory cytokines (il-10 and tgfb1a), and the pro-angiogenic transcription factor (prox1a) (Figures 3C–E). These data indicate that TAN IIA promotes macrophage recruitment and polarization toward the M2 phenotype, thereby facilitating vascular repair in zebrafish.
Figure 3.
TAN IIA enhances macrophage recruitment to sites of vascular injury. (A) TAN IIA promotes the recruitment of macrophages to sites of vascular injury, green-labeled macrophages. (B) Count the number of macrophages within the vascular injury area. (C) Effect of TAN IIA on the mRNA expression levels of mpeg1. (D) Effect of TAN IIA on the mRNA expression levels of mfap4, prox1a. (E) Effect of TAN IIA on mRNA expression levels in M2 macrophages of mrc1a, il-4, tgfb1a, il-10. The relative mRNA expression of candidate genes (as indicated) was assessed by q-PCR after TAN IIA (2 μM) and dmso (2 μM) treatment for 6h (n = 3, three independent experiments). At 6h, the transcriptional level of mpeg1, mfap4, prox1a, mcr1a, il 4, tgfb1a, il 10 were increased. * P < 0.1 versus control group, ** P < 0.01 versus control group, *** P < 0.001 versus control group, **** P < 0.0001 versus control group.
3.4. Identifying potential targets for TAN IIA and vascular injury through network pharmacology analysis
Previous studies have demonstrated that the CXCR4A-CXCL12B signaling axis plays a crucial role in restoring homeostasis in damaged tissues and blood vessels (48). CXCL12, secreted by endothelial cells, is known to promote macrophage migration through activation of CXCR4 (49–51). To further elucidate the role of TAN IIA in vascular injury, we identified relevant targets linking TAN IIA to vascular injury diseases through network pharmacology analysis. We identified MMP9, a core functional gene of M2 macrophages, as a central target in the TAN IIA-vascular injury disease network(Figures 4A–E). Molecular docking results indicate that TAN IIA exhibits strong binding affinity for cxcr4a, cxcl12b, and IL-4, suggesting potential regulatory interactions between TAN IIA and these molecules (Figures 4F, G). We therefore hypothesized that TAN IIA may enhance the secretion of CXCL12B by vascular endothelial cells, thereby activating cxcr4a and facilitating macrophage migration toward sites of vascular injury. To test this hypothesis, we used Tg (flk1: eGFP) and Tg (mpeg1: eGFP) transgenic zebrafish lines and assessed cxcl12b expression in endothelial cells and cxcr4a expression in macrophages at the injury site by fluorescence in situ hybridization combined with antibody staining. TAN IIA treatment group exhibited higher expressions of the cxcl12b and cxcr4a at the injury site compared with DMSO group. These findings indicate that TAN IIA enhances cxcl12b expression in endothelial cells, thereby promoting the recruitment of cxcr4a-expressing macrophages to injured vessels. In addition, qPCR analysis confirmed consistent up-regulation of these genes at the mRNA level (Figures 4H, I). Collectively, these data demonstrate that TAN IIA may promote macrophage recruitment by activating the CXCR4A-CXCL12B signaling axis.
Figure 4.
TAN IIA may promote macrophage recruitment by activating the CXCR4A-CXCL12B signaling axis. (A) Potential Targets for TAN IIA. (B) Unique Targets Associated with Vascular Injury. (C) TAN IIA acts on the core target of vascular injury. (D) Construction of the Protein-Protein Interaction Network for TAN IIA and Vascular Injury Proteins. (E) TAN IIA and Vascular Injury Drug-Target-Disease Network Diagram. (F, G) Molecular Docking of Tanshinone IIA with IL-4, CXCR4A and CXCL12B. (H) TAN IIA promotes macrophage aggregation in damaged blood vessels and enhances cxcr4a expression at sites of vascular injury. Red arrows indicate sites of vascular injury. Effects of TAN IIA (2 μM) and DMSO (2 μM) on cxcr4a mRNA expression levels. (I) TAN IIA promotes repair of damaged blood vessels and induces high expression of cxcl12b at sites of vascular injury. Red arrows indicate sites of vascular injury. Effects of TAN IIA (2 μM) and DMSO (2 μM) on cxcl12b mRNA expression levels. The relative mRNA expression of candidate genes (as indicated) was assessed by q-PCR after TAN IIA (2 μM) and dmso (2 μM) treatment for 6h (n = 3, three independent experiments). ***P < 0.001 versus control group. ****P < 0.0001 versus control group.
3.5. TAN IIA promotes macrophage migration in vitro
To further validate these findings in vitro, we used human umbilical vein endothelial cells (EA.hy926) and murine macrophages (RAW264.7). EA.hy926 cells were first stimulated with LPS (1 µg/L) to mimic vascular inflammation, followed by treatment with increasing concentrations of TAN IIA (0.5, 1, or 2 µM). The conditioned medium (CM) from TAN IIA–treated endothelial cells and applied to Transwell migration assays. TAN IIA-treated CM significantly increased RAW264.7 migration compared with both DMSO and LPS-only controls (Figures 5A, B). Consistently, a wound-healing assay produced similar results (Figure 5D). Furthermore, q-PCR showed that TAN IIA up-regulated LPS-induced CXCL12 expression in EA.hy926 cells (Figure 5C). These findings indicate that TAN IIA stimulates endothelial cells to secrete CXCL12, thereby promoting macrophage migration.
Figure 5.
TAN IIA-treated CM promotes migration of RAW264.7 macrophages. (A) CM obtained from EA. hy926 cells damaged by LPS (1 μg/L) and treated with TAN IIA significantly induced RAW264.7 cell migration. The migration of RAW264.7 cells was analyzed using the Transwell assay, and cells were photographed after 24 hours of treatment with increasing concentrations of LPS (1 μg/L) and TAN IIA (0.5, 1, and 2 μM) (n = 5). (B) Compared with the DMSO control group and the LPS-damaged group, TAN IIA treatment increased the number of migrating cells. Quantitative analysis of the above cell Transwell assay. (C) Effect of TAN IIA on CXCL12 mRNA expression levels in EA. hy926 cells. The relative mRNA expression levels of CXCL12 were determined by q-PCR in EAhy.926 cells treated with LPS (1 μg/L) for 24 hours and then treated with TAN IIA (0.5, 1, and 2 μM) for 24 hours (n = 3, three independent experiments). After 24 hours, the CXCL12 expression level increased in the TAN IIA -treated group. (D) RAW264.7 cells wound healing experiment: After uniformly calculating the cell wound edges using ImageJ software, the white vertical lines indicate the degree of wound healing. ** P < 0.01 versus control group, **** P < 0.0001 compared to the control group.
3.6. TAN IIA promotes repair of damaged blood vessels in zebrafish via the CXCR4A-CXCL12B signaling axis
AMD3100 is an antagonist of CXCR4A that inhibits the CXCR4A-CXCL12B signaling axis (52). SDF-1β is a matrix-derived CXC chemokine that signals through the CXCR4A receptor and upregulates the CXCR4A-CXCL12B signaling axis. In a zebrafish venous injury model, we observed slower vascular repair in the AMD3100 group, while the AMD3100+TAN IIA group accelerated repair compared to the AMD3100 group. Both the SDF-1β group and the SDF-1β+TAN IIA group accelerated vascular repair in zebrafish. Furthermore, the SDF-1β+TAN IIA group resulted in more complete and rapid vascular repair(Figures 6A, B). q-PCR analysis revealed that vascular remodeling-related genes (mmp9, vegfaa, pdgfba, arg1, angpt1) were significantly downregulated in the AMD3100 treatment for 6h, whereas TAN IIA treatment induced their upregulation(Figures 6C, D). These findings indicate that the CXCR4A-CXCL12B signaling axis plays a crucial role in zebrafish vascular repair, and TAN IIA can activate this signaling pathway.
Figure 6.
TAN IIA accelerates vascular repair through the CXCR4A-CXCL12B signaling axis. (A) AMD3100(10 μM) inhibits the repair of damaged blood vessels in zebrafish, while TAN IIA(2 μM) can rescue this phenomenon. (B) SDF-1β(10 μM) can accelerate the repair of damaged blood vessels in zebrafish to a certain extent. The white triangular arrow indicates the repaired blood vessel. (C, D) The effect of AMD3100 (10 μM), AMD3100 (10 μM)+TAN IIA (2 μM), SDF-1β(10 μM) and SDF-1β(10 μM)+TAN IIA (2 μM) on the mRNA expression levels of vegfaa, arg1, pdgfba, and angpt1 genes. * P < 0.1 versus control group, ** P < 0.01 versus control group, *** P < 0.001 versus control group, **** P < 0.0001 versus control group.
3.7. TAN IIA promotes M2 macrophage migration through the CXCR4A-CXCL12B signaling axis
In the previous experimental results, we observed that AMD3100 inhibits the rate of vascular repair in zebrafish. To elucidate this phenomenon, we treated zebrafish with AMD3100, SDF-1β, and TAN IIA after inducing venous vascular injury. The AMD3100 group exhibited reduced macrophage migration at the vascular injury site, while the AMD3100+TAN IIA group showed greater macrophage migration than the AMD3100 group. The SDF-1β+TAN IIA group demonstrated higher macrophage migration than the AMD3100 group(Figures 7A, B). q-PCR analysis revealed that macrophage-associated genes (mpeg1, mfap4 and prox1a) and M2macrophage-associated genes(mrc1a, mmp9, il 4, il 10, tgfb1a)were significantly downregulated in the AMD3100 group, whereas TAN IIA treatment induced their upregulation(Figures 7C–E). Additionally, Western blotting and q-PCR experiments demonstrated that TAN IIA promotes the upregulation of the JAK1/STAT6 pathway (Figures 7F, G). These findings indicate that TAN IIA promotes macrophage migration to sites of vascular injury to accelerate vascular repair in zebrafish via the CXCR4A-CXCL12B signaling axis.
Figure 7.
TAN IIA accelerates the recruitment of M2 macrophages through the CXCR4A-CXCL12B signaling axis. (A) SDF-1β also promotes macrophage recruitment to sites of vascular injury (green-labeled macrophages) to a certain extent. (B) AMD3100 (10 μM) reduces macrophage recruitment to sites of vascular injury, while TAN IIA(2 μM) can rescue this phenomenon. (C) Effects of AMD3100 (10 μM), AMD3100 (10 μM)+TAN IIA (2 μM), SDF-1β(10 μM) and SDF-1β(10 μM)+TAN IIA (2 μM) on mpeg1, mfap4 and prox1a mRNA expression levels. (D, E) Effects of AMD3100 (10 μM), AMD3100 (10 μM)+TAN IIA (2 μM), SDF-1β(10 μM) and SDF-1β(10 μM)+TAN IIA (2 μM) on mrc1a, il 4, il 10, mmp9 and tgfb1a mRNA expression levels. (F) Effects of AMD3100 (10 μM), AMD3100 (10 μM)+TAN IIA (2 μM), SDF-1β(10 μM) and SDF-1β(10 μM)+TAN IIA (2 μM) on jak1 and stat6 mRNA expression levels. (G) Effects of AMD3100 (10 μM), AMD3100 (10 μM)+TAN IIA (2 μM), SDF-1β(10 μM) on the expression levels of Jak1 and Stat6 proteins. Relative mRNA expression levels of candidate genes (as shown in the figure) were measured by q-PCR 6 hours after treatment with AMD3100 (10 μM), AMD3100 (10 μM)+TAN IIA (2 μM), SDF-1β(10 μM) and SDF-1β(10 μM)+TAN IIA (2 μM) (n=3, three independent experiments). *P < 0.1 versus control group, **P < 0.01 versus control group, ***P < 0.001 versus control group, ****P < 0.0001 versus control group.
3.8. TAN IIA promotes macrophage migration through the CXCR4A-CXCL12B signaling axis in vitro
To validate the role of AMD3100 in in vitro murine macrophages (RAW264.7) migration, we demonstrated through Transwell migration assays that AMD3100 inhibited macrophage migration, and TAN IIA could rescue this effect (Figures 8A, B). Wound healing assays confirmed identical results(Figure 8C). These findings confirm that TAN IIA can rescue the inhibition of macrophage migration induced by AMD3100.
Figure 8.
TAN IIA promotes macrophage migration through the CXCR4A-CXCL12B signaling axis. (A) Conditioned medium (CM) obtained from EA. hy926 cells injured by LPS (1 μg/L) significantly inhibited the migration of RAW264.7 cells after 24-hour treatment with AMD3100. Transwell assays analyzed RAW264.7 cell migration, with images captured after 24-hour treatment in different CM groups (n=5). Compared to the DMSO control and LPS-injured groups, AMD3100 treatment significantly suppressed the number of migrating cells, while TAN IIA could rescue this effect. (B) Quantitative analysis of the cell Transwell assay. (C) Wound healing assay in RAW264.7 cells: After uniformly calculating the cell wound edges using ImageJ software, the white vertical line indicates the extent of wound healing. * P < 0.1 versus control group, ** P < 0.01 versus control group, ****P < 0.0001 versus control group.
3.9. TAN IIA promotes vascular repair in zebrafish via the CXCR4A-CXCL12B signaling axis
Two-photon infrared laser–induced vessel injury experiments (Figure 9) show that TAN IIA stimulates endothelial cells to secrete cxcl12b, which activates cxcr4a in macrophages. Activation of this signaling pathway promotes macrophage recruitment and M2 polarization, accelerating the repair of injured blood vessels in zebrafish.
Figure 9.
Mechanism diagram of TAN IIA promoting repair of damaged blood vessels. TAN IIA treatment enhances chemokine CXCL12B secretion by endothelial cells, thereby increasing macrophage recruitment via activation of the CXCR4A-CXCL12B signaling axis and ultimately facilitating the repair of damaged blood vessels in zebrafish.
4. Discussion
Salvia miltiorrhiza Bunge, a perennial plant of the Labiatae family (8), possesses diverse biological activities, including antioxidant, antibacterial, and anti-inflammatory effects. Owing to these properties, it has been widely used in the treatment of cardiovascular and cerebrovascular diseases (9). TAN IIA, a fat-soluble diterpene quinone and the principal bioactive component of S. miltiorrhiza, exhibits multiple cardiovascular and cerebrovascular benefits. It can inhibit oxidative stress and inflammatory injury in vascular endothelial cells, thereby improving endothelial dysfunction, and protects vascular smooth muscle cells by suppressing their proliferation and migration, which reduces vascular stenosis (11). However, its therapeutic effects in the context of vascular injury remain incompletely characterized, and its potential role in vascular repair has not been fully explored.
In this study, we demonstrate for the first time that TAN IIA promotes vascular repair in a laser-induced zebrafish vein injury model. Its mechanism of action involves enhancing macrophage recruitment by activating the CXCR4A-CXCL12B signaling axis and accelerating vascular repair through M2 macrophages.
Cardiovascular disease has become a major global health challenge, and vascular integrity is a critical determinant of disease onset and progression. Pathological changes in blood vessels are central to many cardiovascular disorders. Previous work has shown that TAN IIA can help prevent vascular dysfunction (11). Building on these findings, our results reveal that TAN IIA markedly enhances repair of infrared-laser-induced venous injury in zebrafish. Moreover, the up-regulation of vascular remodeling genes such as arg1, mmp9, vegfaa, pdgfba, and angpt1 (51, 53–56) suggests that TAN IIA promotes vascular regeneration at the molecular level.
Macrophages are multifunctional immune cells that play pivotal roles in tissue and vascular remodeling (14–16). Recent studies have highlighted their importance in promoting vascular regeneration (13). Consistent with these findings, our results show that TAN IIA induces macrophage accumulation at sites of vascular injury and up-regulates macrophage-related genes. In vitro, conditioned medium from TAN IIA–treated EA.hy926 endothelial cells enhanced RAW264.7 macrophage migration, accompanied by increased CXCL12 expression, indicating that endothelial-derived CXCL12 mediates TAN IIA–induced macrophage chemotaxis. M2 macrophages—also referred to as anti-inflammatory macrophages—express MRC1 as a surface marker (57) and can be induced by the immune regulatory cytokine IL-4 (18, 19). These cells produce anti-inflammatory mediators, such as IL-10 and TGF-β, which contribute to vascular repair and homeostasis (22, 23, 27, 29). Additional markers of macrophage identity and function include mpeg1 and mfap4, which are also involved in hematopoiesis and myeloid cell development (58, 59). Notably, mfap4 promotes vascular smooth-muscle proliferation and neovascularization (60), while prox1a supports late-stage vascular remodeling (43). Our study revealed that TAN IIA significantly upregulates the expression of il 4, mrc1a, il 10, and tgfb1a indicating that it promotes vascular repair by enhancing il 10 and tgfb1a secretion through M2 macrophages. Concurrently, TAN IIA also increased the expression of mfap4 and prox1a, further confirming its role in vascular remodeling.
The CXCR4/CXCL12 signaling axis is known to guide macrophage migration toward injured vessels. CXCL12, secreted by endothelial cells, mediates unidirectional migration of mononuclear macrophages through CXCR4 activation (49, 50, 61). Consistent with this pathway, we observed that TAN IIA up-regulated cxcr4a and cxcl12b expression at the injury site, and enhanced CXCL12 expression in EA.hy926 cells. These findings suggest that TAN IIA promotes macrophage recruitment to damaged vessels via the CXCR4A-CXCL12B axis.
To further validate this finding, we treated zebrafish with vascular injuries using AMD3100 and SDF-1β. AMD3100 is a CXCR4A antagonist (52), while SDF-1β promotes the migration and infiltration of macrophages expressing CXCR4A (62). We found that AMD3100 inhibited macrophage migration to the site of vascular injury in zebrafish and suppressed repair of damaged blood vessels, whereas TAN IIA reversed this effect. Similar results were obtained in in vitro experiments using RAW264.7 cells. Furthermore, we observed that SDF-1β promotes macrophage migration to injured zebrafish blood vessels and accelerates vascular repair, while TAN IIA exhibits a stronger angiogenic effect. Network pharmacology analysis revealed that MMP9, a core functional gene of M2 macrophages, plays a crucial role in TAN IIA and vascular injury. Molecular docking experiments revealed strong binding affinity between TAN IIA and IL4, CXCR4A, and CXCL12B. Furthermore, Western blotting and q-PCR experiments demonstrated that TAN IIA promotes upregulation of the JAK1/STAT6 pathway and enhances the expression of M2 macrophage-related genes (mrc1a, mmp9, il 4, il 10, tgfb1a). We hypothesize that TAN IIA may also promote macrophage conversion to M2 macrophages, though the mechanism remains unexplored. We propose that TAN IIA accelerates the repair of damaged blood vessels in zebrafish via M2-type macrophages.
This study confirms that TAN IIA accelerates the repair process of zebrafish venous vessels by recruiting macrophages through the CXCR4A-CXCL12B signaling axis. It is known that endothelial nitric oxide synthase (eNOS) serves as a key molecular target for TAN IIA ‘s vascular regulatory effects (63). Previous studies have demonstrated that TAN IIA plays a crucial role in the prevention and treatment of cardiovascular diseases by activating the eNOS pathway (64, 65). However, no direct experimental evidence currently exists to confirm a direct link between TAN IIA ‘s regulation of eNOS and the specific process of macrophage recruitment. Based on this, we hypothesize that TAN IIA may indirectly facilitate CXCL12B-mediated macrophage repair of damaged vessels by regulating eNOS activity in vascular endothelial cells, thereby maintaining vascular integrity and stabilizing the local microenvironment. Future experimental validation of the specific role of the eNOS pathway in TAN IIA ‘s regulation of macrophage recruitment will be a key subsequent research direction for elucidating the molecular mechanism by which TAN IIA treats vascular injury through macrophage recruitment.
In summary, our results identify macrophage recruitment as a key driver of rapid vascular repair in zebrafish. TAN IIA accelerates the repair of damaged zebrafish blood vessels by promoting M2 macrophage recruitment through activation of the CXCR4A-CXCL12B signaling pathway. As a widely used cardiovascular agent derived from Salvia miltiorrhiza Bunge, TAN IIA shows strong potential as a therapeutic candidate for vascular injury and regeneration.
Funding Statement
The author(s) declared that financial support was received for this work and/or its publication. This project is supported by the National Natural Science Foundation of China (32160168, 32260177, 32360214), and in part by the Guizhou Province’s Science and Technology Major Project (GCC[2023]034, ZK[2024]-143), and the Guizhou Medical University Key Laboratory Project ([2024]004) and the Qiankehe Platform, Project No. KXJZ[2024]004.
Edited by: Carolina Otero, Universidad Andrés Bello, Chile
Reviewed by: Raghavan Chinnadurai, Mercer University, United States
Songfeng Hu, Shaoxing Hospital of Traditional Chinese Medicine, China
Abbreviations: CXCL12, C-X-C motif chemokine ligand 12; mpeg1, macrophage expressed 1; TAN IIA, Tanshinone IIA; mfap4, microfibril associated protein 4; mmp9, matrix metallopeptidase 9; prox1a, prospero homeobox 1a; pdgfba, platelet-derived growth factor beta polypeptide a; tgfb1a, transforming growth factor, beta 1a; il-10, interleukin 10; angpt1, angiopoietin 1; vegfaa, vascular endothelial growth factor Aa; il-4, interleukin 4; cxcl12b, chemokine (C-X-C motif) ligand 12b; mrc1a, mannose receptor, C type 1a; cxcr4a, chemokine (C-X-C motif) receptor 4a; jak1, Janus kinase 1; arg1, arginase 1; stat6, signal transducer and activator of transcription 6.
Data availability statement
The original contributions presented in the study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding authors.
Ethics statement
The animal study was approved by Guizhou Medical University the Animal Care Welfare Committee. The study was conducted in accordance with the local legislation and institutional requirements.
Author contributions
LZ: Validation, Investigation, Formal Analysis, Methodology, Data curation, Writing – original draft. LA: Investigation, Data curation, Writing – review & editing, Formal Analysis, Methodology. YZ: Writing – review & editing, Methodology, Investigation, Data curation. ZL: Investigation, Writing – review & editing, Methodology. JH: Writing – review & editing, Investigation, Methodology. LT: Methodology, Investigation, Writing – review & editing. ML: Writing – review & editing, Investigation, Methodology. DM: Methodology, Writing – review & editing, Investigation, Formal Analysis, Funding acquisition, Project administration. LS: Writing – review & editing, Funding acquisition, Conceptualization, Project administration, Supervision.
Conflict of interest
The author(s) declared that this work was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Generative AI statement
The author(s) declared that generative AI was not used in the creation of this manuscript.
Any alternative text (alt text) provided alongside figures in this article has been generated by Frontiers with the support of artificial intelligence and reasonable efforts have been made to ensure accuracy, including review by the authors wherever possible. If you identify any issues, please contact us.
Publisher’s note
All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.
Supplementary material
The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fimmu.2026.1792022/full#supplementary-material
References
- 1. Padro T, Manfrini O, Bugiardini R, Canty J, Cenko E, De Luca G, et al. ESC Working Group on Coronary Pathophysiology and Microcirculation position paper on ‘coronary microvascular dysfunction in cardiovascular disease’. Cardiovasc Res. (2020) 116:741–55. doi: 10.1093/cvr/cvaa003, PMID: [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2. Chu Q, Li Y, Wu J, Gao Y, Guo X, Li J, et al. Oxysterol sensing through GPR183 triggers endothelial senescence in hypertension. Circ Res. (2024) 135:708–21. doi: 10.1161/CIRCRESAHA.124.324722, PMID: [DOI] [PubMed] [Google Scholar]
- 3. Monteiro JP, Bennett M, Rodor J, Caudrillier A, Ulitsky I, Baker AH. Endothelial function and dysfunction in the cardiovascular system: the long non-coding road. Cardiovasc Res. (2019) 115:1692–704. doi: 10.1093/cvr/cvz154, PMID: [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4. Chen F, Li L, Tian DD. Salvia miltiorrhiza Roots against Cardiovascular Disease: Consideration of Herb-Drug Interactions. BioMed Res Int. (2017) 2017:9868694. doi: 10.1155/2017/9868694, PMID: [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5. Ponikowski P, Voors AA, Anker SD, Bueno H, Cleland JGF, Coats AJS, et al. 2016 ESC Guidelines for the diagnosis and treatment of acute and chronic heart failure: The Task Force for the diagnosis and treatment of acute and chronic heart failure of the European Society of Cardiology (ESC)Developed with the special contribution of the Heart Failure Association (HFA) of the ESC. Eur Heart J. (2016) 37:2129–200. doi: 10.1093/eurheartj/ehw128, PMID: [DOI] [PubMed] [Google Scholar]
- 6. Collins R, Reith C, Emberson J, Armitage J, Baigent C, Blackwell L, et al. Interpretation of the evidence for the efficacy and safety of statin therapy. Lancet. (2016) 388:2532–61. doi: 10.1016/S0140-6736(16)31357-5, PMID: [DOI] [PubMed] [Google Scholar]
- 7. Yang Z, Lin S, Liu Y, Ren Q, Ge Z, Wang C, et al. Traditional chinese medicine in coronary microvascular disease. Front Pharmacol. (2022) 13:929159. doi: 10.3389/fphar.2022.929159, PMID: [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8. Zeng H, Su S, Xiang X, Sha X, Zhu Z, Wang Y, et al. Comparative Analysis of the Major Chemical Constituents in Salvia miltiorrhiza Roots, Stems, Leaves and Flowers during Different Growth Periods by UPLC-TQ-MS/MS and HPLC-ELSD Methods. Molecules. (2017) 22:771. doi: 10.3390/molecules22050771, PMID: [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9. Chong CM, Su H, Lu JJ, Wang Y. The effects of bioactive components from the rhizome of Salvia miltiorrhiza (Danshen) on the characteristics of Alzheimer’s disease. Chin Med. (2019) 14:19. doi: 10.1186/s13020-019-0242-0, PMID: [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10. Jia LQ, Yang GL, Ren L, Chen WN, Feng JY, Cao Y, et al. Tanshinone IIA reduces apoptosis induced by hydrogen peroxide in the human endothelium-derived EA.hy926 cells. J Ethnopharmacol. (2012) 143:100–8. doi: 10.1016/j.jep.2012.06.007, PMID: [DOI] [PubMed] [Google Scholar]
- 11. Yang C, Mu Y, Li S, Zhang Y, Liu X, Li J. Tanshinone IIA: a Chinese herbal ingredient for the treatment of atherosclerosis. Front Pharmacol. (2023) 14:1321880. doi: 10.3389/fphar.2023.1321880, PMID: [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12. Wang J, Zhang Y, Feng X, Du M, Li S, Chang X, et al. Tanshinone IIA alleviates atherosclerosis in LDLR(-/-) mice by regulating efferocytosis of macrophages. Front Pharmacol. (2023) 14:1233709. doi: 10.3389/fphar.2023.1233709, PMID: [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13. Yan L, Wang J, Cai X, Liou YC, Shen HM, Hao J, et al. Macrophage plasticity: signaling pathways, tissue repair, and regeneration. MedComm (2020). (2024) 5:e658. doi: 10.1002/mco2.658, PMID: [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14. Hassanshahi A, Moradzad M, Ghalamkari S, Fadaei M, Cowin AJ, Hassanshahi M. Macrophage-mediated inflammation in skin wound healing. Cells. (2022) 11:2953. doi: 10.3390/cells11192953, PMID: [DOI] [PMC free article] [PubMed] [Google Scholar]
- 15. Kloc M, Ghobrial RM, Wosik J, Lewicka A, Lewicki S, Kubiak JZ. Macrophage functions in wound healing. J Tissue Eng Regener Med. (2019) 13:99–109. doi: 10.1002/term.2772, PMID: [DOI] [PubMed] [Google Scholar]
- 16. Raziyeva K, Kim Y, Zharkinbekov Z, Kassymbek K, Jimi S, Saparov A. Immunology of acute and chronic wound healing. Biomolecules. (2021) 11:700. doi: 10.3390/biom11050700, PMID: [DOI] [PMC free article] [PubMed] [Google Scholar]
- 17. Wynn TA, Vannella KM. Macrophages in tissue repair, regeneration, and fibrosis. Immunity. (2016) 44:450–62. doi: 10.1016/j.immuni.2016.02.015, PMID: [DOI] [PMC free article] [PubMed] [Google Scholar]
- 18. Gao S, Zhou J, Liu N, Wang L, Gao Q, Wu Y, et al. Curcumin induces M2 macrophage polarization by secretion IL-4 and/or IL-13. J Mol Cell Cardiol. (2015) 85:131–9. doi: 10.1016/j.yjmcc.2015.04.025, PMID: [DOI] [PubMed] [Google Scholar]
- 19. He Y, Gao Y, Zhang Q, Zhou G, Cao F, Yao S. IL-4 switches microglia/macrophage M1/M2 polarization and alleviates neurological damage by modulating the JAK1/STAT6 pathway following ICH. Neuroscience. (2020) 437:161–71. doi: 10.1016/j.neuroscience.2020.03.008, PMID: [DOI] [PubMed] [Google Scholar]
- 20. Huang X, Li Y, Fu M, Xin HB. Polarizing macrophages in vitro. Methods Mol Biol. (2018) 1784:119–26. doi: 10.1007/978-1-4939-7837-3_12, PMID: [DOI] [PMC free article] [PubMed] [Google Scholar]
- 21. Lundahl MLE, Mitermite M, Ryan DG, Case S, Williams NC, Yang M, et al. Macrophage innate training induced by IL-4 and IL-13 activation enhances OXPHOS driven anti-mycobacterial responses. Elife. (2022) 11:e74690. doi: 10.7554/eLife.74690.sa2, PMID: [DOI] [PMC free article] [PubMed] [Google Scholar]
- 22. Fang J, Ou Q, Wu B, Li S, Wu M, Qiu J, et al. TcpC Inhibits M1 but Promotes M2 Macrophage Polarization via Regulation of the MAPK/NF-κB and Akt/STAT6 Pathways in Urinary Tract Infection. Cells. (2022) 11:2674. doi: 10.3390/cells11172674, PMID: [DOI] [PMC free article] [PubMed] [Google Scholar]
- 23. Wang J, Xu L, Xiang Z, Ren Y, Zheng X, Zhao Q, et al. Microcystin-LR ameliorates pulmonary fibrosis via modulating CD206(+) M2-like macrophage polarization. Cell Death Dis. (2020) 11:136. doi: 10.1038/s41419-020-2329-z, PMID: [DOI] [PMC free article] [PubMed] [Google Scholar]
- 24. Xu ZJ, Gu Y, Wang CZ, Jin Y, Wen XM, Ma JC, et al. The M2 macrophage marker CD206: a novel prognostic indicator for acute myeloid leukemia. Oncoimmunology. (2020) 9:1683347. doi: 10.1080/2162402X.2019.1683347, PMID: [DOI] [PMC free article] [PubMed] [Google Scholar]
- 25. Zhu Y, Sun X, Tan S, Luo C, Zhou J, Zhang S, et al. M2 macrophage-related gene signature in chronic rhinosinusitis with nasal polyps. Front Immunol. (2022) 13:1047930. doi: 10.3389/fimmu.2022.1047930, PMID: [DOI] [PMC free article] [PubMed] [Google Scholar]
- 26. Cai G, Lu Y, Zhong W, Wang T, Li Y, Ruan X, et al. Piezo1-mediated M2 macrophage mechanotransduction enhances bone formation through secretion and activation of transforming growth factor-β1. Cell Prolif. (2023) 56:e13440. doi: 10.1111/cpr.13440, PMID: [DOI] [PMC free article] [PubMed] [Google Scholar]
- 27. Chen X, Wan Z, Yang L, Song S, Fu Z, Tang K, et al. Exosomes derived from reparative M2-like macrophages prevent bone loss in murine periodontitis models via IL-10 mRNA. J Nanobiotechnology. (2022) 20:110. doi: 10.1186/s12951-022-01314-y, PMID: [DOI] [PMC free article] [PubMed] [Google Scholar]
- 28. Fu XH, Li JP, Li XY, Tan Y, Zhao M, Zhang SF, et al. M2-macrophage-derived exosomes promote meningioma progression through TGF-β Signaling pathway. J Immunol Res. (2022) 2022:8326591. doi: 10.1155/2022/8326591, PMID: [DOI] [PMC free article] [PubMed] [Google Scholar]
- 29. Hu J, Deng F, Zhao B, Lin Z, Sun Q, Yang X, et al. Lactobacillus murinus alleviate intestinal ischemia/reperfusion injury through promoting the release of interleukin-10 from M2 macrophages via Toll-like receptor 2 signaling. Microbiome. (2022) 10:38. doi: 10.1186/s40168-022-01227-w, PMID: [DOI] [PMC free article] [PubMed] [Google Scholar]
- 30. Li S, Ding X, Zhang H, Ding Y, Tan Q. IL-25 improves diabetic wound healing through stimulating M2 macrophage polarization and fibroblast activation. Int Immunopharmacol. (2022) 106:108605. doi: 10.1016/j.intimp.2022.108605, PMID: [DOI] [PubMed] [Google Scholar]
- 31. Oh S, Lee JH, Kim HM, Batsukh S, Sung MJ, Lim TH, et al. Poly-L-lactic acid fillers improved dermal collagen synthesis by modulating M2 macrophage polarization in aged animal skin. Cells. (2023) 12:1320. doi: 10.3390/cells12091320, PMID: [DOI] [PMC free article] [PubMed] [Google Scholar]
- 32. Wang Y, Geng X, Guo Z, Chu D, Liu R, Cheng B, et al. M2 macrophages promote subconjunctival fibrosis through YAP/TAZ signalling. Ann Med. (2024) 56:2313680. doi: 10.1080/07853890.2024.2313680, PMID: [DOI] [PMC free article] [PubMed] [Google Scholar]
- 33. Aitcheson SM, Frentiu FD, Hurn SE, Edwards K, Murray RZ. Skin wound healing: normal macrophage function and macrophage dysfunction in diabetic wounds. Molecules. (2021) 26:4917. doi: 10.3390/molecules26164917, PMID: [DOI] [PMC free article] [PubMed] [Google Scholar]
- 34. Govindappa PK, Elfar JC. Erythropoietin promotes M2 macrophage phagocytosis of Schwann cells in peripheral nerve injury. Cell Death Dis. (2022) 13:245. doi: 10.1038/s41419-022-04671-6, PMID: [DOI] [PMC free article] [PubMed] [Google Scholar]
- 35. Razi S, Yaghmoorian Khojini J, Kargarijam F, Panahi S, Tahershamsi Z, Tajbakhsh A, et al. Macrophage efferocytosis in health and disease. Cell Biochem Funct. (2023) 41:152–65. doi: 10.1002/cbf.3780, PMID: [DOI] [PubMed] [Google Scholar]
- 36. Zhong X, Lee HN, Kim SH, Park SA, Kim W, Cha YN, et al. Myc-nick promotes efferocytosis through M2 macrophage polarization during resolution of inflammation. FASEB J. (2018) 32:5312–25. doi: 10.1096/fj.201800223R, PMID: [DOI] [PubMed] [Google Scholar]
- 37. Li X, Ren Y, Chang K, Wu W, Griffiths HR, Lu S, et al. Adipose tissue macrophages as potential targets for obesity and metabolic diseases. Front Immunol. (2023) 14:1153915. doi: 10.3389/fimmu.2023.1153915, PMID: [DOI] [PMC free article] [PubMed] [Google Scholar]
- 38. Russo S, Kwiatkowski M, Govorukhina N, Bischoff R, Melgert BN. Meta-inflammation and metabolic reprogramming of macrophages in diabetes and obesity: the importance of metabolites. Front Immunol. (2021) 12:746151. doi: 10.3389/fimmu.2021.746151, PMID: [DOI] [PMC free article] [PubMed] [Google Scholar]
- 39. Ying W, Gao H, Dos Reis FCG, Bandyopadhyay G, Ofrecio JM, Luo Z, et al. MiR-690, an exosomal-derived miRNA from M2-polarized macrophages, improves insulin sensitivity in obese mice. Cell Metab. (2021) 33:781–90.e5. doi: 10.1016/j.cmet.2020.12.019, PMID: [DOI] [PMC free article] [PubMed] [Google Scholar]
- 40. Li S, Yang P, Ding X, Zhang H, Ding Y, Tan Q. Puerarin improves diabetic wound healing via regulation of macrophage M2 polarization phenotype. Burns Trauma. (2022) 10:tkac046. doi: 10.1093/burnst/tkac046, PMID: [DOI] [PMC free article] [PubMed] [Google Scholar]
- 41. Pi L, Fang B, Meng X, Qian L. LncRNA XIST accelerates burn wound healing by promoting M2 macrophage polarization through targeting IL-33 via miR-19b. Cell Death Discov. (2022) 8:220. doi: 10.1038/s41420-022-00990-x, PMID: [DOI] [PMC free article] [PubMed] [Google Scholar]
- 42. Savitri C, Kwon JW, Drobyshava V, Ha SS, Park K. M2 macrophage-derived concentrated conditioned media significantly improves skin wound healing. Tissue Eng Regener Med. (2022) 19:617–28. doi: 10.1007/s13770-021-00414-4, PMID: [DOI] [PMC free article] [PubMed] [Google Scholar]
- 43. Bai H, Xie B, Li M, Sun P, Wei S, Zhang L, et al. Biodegraded PCl and gelatin fabricated vascular patch in rat aortic and inferior vena cava angioplasty. Microvasc Res. (2022) 141:104314. doi: 10.1016/j.mvr.2022.104314, PMID: [DOI] [PubMed] [Google Scholar]
- 44. Yan D, He Y, Dai J, Yang L, Wang X, Ruan Q. Vascular endothelial growth factor modified macrophages transdifferentiate into endothelial-like cells and decrease foam cell formation. Biosci Rep. (2017) 37:BSR20170002. doi: 10.1042/BSR20170002, PMID: [DOI] [PMC free article] [PubMed] [Google Scholar]
- 45. Liu C, Wu C, Yang Q, Gao J, Li L, Yang D, et al. Macrophages mediate the repair of brain vascular rupture through direct physical adhesion and mechanical traction. Immunity. (2016) 44:1162–76. doi: 10.1016/j.immuni.2016.03.008, PMID: [DOI] [PubMed] [Google Scholar]
- 46. He J, Mo D, Chen J, Luo L. Combined whole-mount fluorescence in situ hybridization and antibody staining in zebrafish embryos and larvae. Nat Protoc. (2020) 15:3361–79. doi: 10.1038/s41596-020-0376-7, PMID: [DOI] [PubMed] [Google Scholar]
- 47. Wang T, Wang C, Wu Q, Zheng K, Chen J, Lan Y, et al. Evaluation of tanshinone IIA developmental toxicity in zebrafish embryos. Molecules. (2017) 22. doi: 10.3390/molecules22040660, PMID: [DOI] [PMC free article] [PubMed] [Google Scholar]
- 48. Isles HM, Herman KD, Robertson AL, Loynes CA, Prince LR, Elks PM, et al. The CXCL12/CXCR4 signaling axis retains neutrophils at inflammatory sites in zebrafish. Front Immunol. (2019) 10:1784. doi: 10.3389/fimmu.2019.01784, PMID: [DOI] [PMC free article] [PubMed] [Google Scholar]
- 49. Arwert EN, Harney AS, Entenberg D, Wang Y, Sahai E, Pollard JW, et al. A unidirectional transition from migratory to perivascular macrophage is required for tumor cell intravasation. Cell Rep. (2018) 23:1239–48. doi: 10.1016/j.celrep.2018.04.007, PMID: [DOI] [PMC free article] [PubMed] [Google Scholar]
- 50. Mai CL, Tan Z, Xu YN, Zhang JJ, Huang ZH, Wang D, et al. CXCL12-mediated monocyte transmigration into brain perivascular space leads to neuroinflammation and memory deficit in neuropathic pain. Theranostics. (2021) 11:1059–78. doi: 10.7150/thno.44364, PMID: [DOI] [PMC free article] [PubMed] [Google Scholar]
- 51. Bernocchi F, Bonomi CG, Assogna M, Moreschini A, Mercuri NB, Koch G, et al. Astrocytic-derived vascular remodeling factors are independently associated with blood brain barrier permeability in Alzheimer’s disease. Neurobiol Aging. (2024) 141:66–73. doi: 10.1016/j.neurobiolaging.2024.05.002, PMID: [DOI] [PubMed] [Google Scholar]
- 52. Evans MO, Smith DM, Kress AT, Nadeau RJ, Selig DJ, Caridha D, et al. Plerixafor for pathogen-agnostic treatment in murine thigh infection and zebrafish sepsis. Clin Transl Sci. (2024) 17:e13876. doi: 10.1111/cts.13876, PMID: [DOI] [PMC free article] [PubMed] [Google Scholar]
- 53. Di Y, Nie QZ, Chen XL. Matrix metalloproteinase-9 and vascular endothelial growth factor expression change in experimental retinal neovascularization. Int J Ophthalmol. (2016) 9:804–8. doi: 10.18240/ijo.2016.06.02, PMID: [DOI] [PMC free article] [PubMed] [Google Scholar]
- 54. Khan A, Parray A, Akhtar N, Agouni A, Kamran S, Pananchikkal SV, et al. Corneal nerve loss in patients with TIA and acute ischemic stroke in relation to circulating markers of inflammation and vascular integrity. Sci Rep. (2022) 12:3332. doi: 10.1038/s41598-022-07353-7, PMID: [DOI] [PMC free article] [PubMed] [Google Scholar]
- 55. Fujiwara-Sumiyoshi S, Ueda Y, Fujikawa M, Osaki M, Yamanaka N, Matsumoto T. Mucopolysaccharide polysulfate promotes microvascular stabilization and barrier integrity of dermal microvascular endothelial cells via activation of the angiopoietin-1/Tie2 pathway. J Dermatol Sci. (2021) 103:25–32. doi: 10.1016/j.jdermsci.2021.05.008, PMID: [DOI] [PubMed] [Google Scholar]
- 56. Hori Y, Kitani T, Yanishi K, Suga T, Kogure M, Kusaba T, et al. Intravenous administration of human Muse cells recovers blood flow in a mouse model of hindlimb ischemia. Front Cardiovasc Med. (2022) 9:981088. doi: 10.3389/fcvm.2022.981088, PMID: [DOI] [PMC free article] [PubMed] [Google Scholar]
- 57. Nasim S, Bichsel C, Dayneka S, Mannix R, Holm A, Vivero M, et al. MRC1 and LYVE1 expressing macrophages in vascular beds of GNAQ p.R183Q driven capillary malformations in Sturge Weber syndrome. Acta Neuropathol Commun. (2024) 12:47. doi: 10.1186/s40478-024-01757-4, PMID: [DOI] [PMC free article] [PubMed] [Google Scholar]
- 58. Mohammadi A, Sorensen GL, Pilecki B. MFAP4-mediated effects in elastic fiber homeostasis, integrin signaling and cancer, and its role in teleost fish. Cells. (2022) 11. doi: 10.3390/cells11132115, PMID: [DOI] [PMC free article] [PubMed] [Google Scholar]
- 59. Kaveh A, Bruton FA, Buckley C, Oremek MEM, Tucker CS, Mullins JJ, et al. Live imaging of heart injury in larval zebrafish reveals a multi-stage model of neutrophil and macrophage migration. Front Cell Dev Biol. (2020) 8:579943. doi: 10.3389/fcell.2020.579943, PMID: [DOI] [PMC free article] [PubMed] [Google Scholar]
- 60. Schlosser A, Pilecki B, Hemstra LE, Kejling K, Kristmannsdottir GB, Wulf-Johansson H, et al. MFAP4 promotes vascular smooth muscle migration, proliferation and accelerates neointima formation. Arterioscler Thromb Vasc Biol. (2016) 36:122–33. doi: 10.1161/ATVBAHA.115.306672, PMID: [DOI] [PubMed] [Google Scholar]
- 61. Döring Y, van der Vorst EPC, Duchene J, Jansen Y, Gencer S, Bidzhekov K, et al. CXCL12 derived from endothelial cells promotes atherosclerosis to drive coronary artery disease. Circulation. (2019) 139:1338–40. doi: 10.1161/CIRCULATIONAHA.118.037953, PMID: [DOI] [PMC free article] [PubMed] [Google Scholar]
- 62. Janowski M. Functional diversity of SDF-1 splicing variants. Cell Adhesion Migration. (2009) 3:243–9. doi: 10.4161/cam.3.3.8260, PMID: [DOI] [PMC free article] [PubMed] [Google Scholar]
- 63. Kim DD, Sánchez FA, Durán RG, Kanetaka T, Durán WN. Endothelial nitric oxide synthase is a molecular vascular target for the Chinese herb Danshen in hypertension. Am J Physiol Heart Circ Physiol. (2007) 292:H2131–7. doi: 10.1152/ajpheart.01027.2006, PMID: [DOI] [PubMed] [Google Scholar]
- 64. Chan P, Liu JC, Lin LJ, Chen PY, Cheng TH, Lin JG, et al. Tanshinone IIA inhibits angiotensin II-induced cell proliferation in rat cardiac fibroblasts. Am J Chin Med. (2011) 39:381–94. doi: 10.1142/S0192415X11008890, PMID: [DOI] [PubMed] [Google Scholar]
- 65. Wang P, Gu Y, Lu J, Song M, Hou W, Li P, et al. Endothelial TRPV4 channel mediates the vasodilation induced by Tanshinone IIA. Chem Biol Interact. (2024) 402:111181. doi: 10.1016/j.cbi.2024.111181, PMID: [DOI] [PubMed] [Google Scholar]
Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Data Availability Statement
The original contributions presented in the study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding authors.