Shuxuening injection improves ventricular remodeling after myocardial infarction by indirectly promoting macrophage-mediated angiogenesis and lymphangiogenesis
Liuqing Yang, Xiaoshuai Zhang, Yajuan Zheng, He Wang, Mingliang Zhang, Yali Wu, Hui Zhang, Xiaoyan Wang, Yingjie Cao, Pan Wang, Bin Wang, Shiting Wei, Yuhui Li, Weixia Li, Xiaofei Chen, Jinfa Tang
TL;DR
Shuxuening injection helps repair heart damage after heart attacks by boosting blood and lymph vessel growth through macrophages.
Contribution
The study reveals that SXNI indirectly promotes angiogenesis and lymphangiogenesis via macrophage activation, identifying key active compounds.
Findings
SXNI improves heart function and reduces infarct size in rats after myocardial infarction.
SXNI activates VEGF-A/VEGFR2 and VEGF-C/VEGFR3 pathways, promoting angiogenesis and lymphangiogenesis.
Ginkgolide A, B, rutin, and quercetin 3-neohesperidoside are key SXNI components that regulate VEGF expression in macrophages.
Abstract
Macrophage-mediated angiogenesis and lymphangiogenesis after myocardial infarction (MI) are essential for restoring cardiac perfusion and lymphatic drainage, thereby limiting cardiac tissue ischemia, edema, and fibrosis. Shuxuening injection (SXNI) is commonly used in the treatment of cardiovascular diseases in clinical practice, but its mechanism of action in mitigating cardiac remodeling after MI is still unclear. This study aimed to investigate the effect of SXNI on ventricular remodeling after MI and to clarify its mechanism of action. SXNI were administered at doses of 1.05 (low-dose), 2.1 (clinical equivalent-dose), and 4.2 (high-dose) mL/kg/day over a 4-week period by using a rat MI model. Pharmacodynamic assessments encompassed cardiac function, infarct size, and fibrosis areas. Angiogenesis and lymphangiogenesis were assessed via immunohistochemical staining, western…
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Figure 13- —National Natural Science Foundation of China
- —Henan Province Chinese Medicine Scientific Research Special Project
- —Medical Science and Technology Project of. Henan Province
- —Key project of Henan Provincial Medical Science and Technology Research Plan
- —Henan Provincial Health Commission National Clinical research base of traditional Chinese medicine research project
- —Science and Technology Innovation Team in Universities of Henan Province
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Taxonomy
TopicsLymphatic System and Diseases · Cardiac Fibrosis and Remodeling · Tissue Engineering and Regenerative Medicine
Introduction
Myocardial infarction (MI) is a prevalent cardiovascular condition characterized by substantial morbidity and mortality, triggered by the blockage of a coronary artery, resulting in impaired cardiomyocyte function and diminished heart performance [1]. Current treatment modalities for MI include thrombolysis, percutaneous coronary intervention (PCI), and coronary artery bypass grafting (CABG) [2–4]. However, reperfusion itself can paradoxically induce cardiomyocyte damage, diminishing therapeutic effectiveness and exacerbating myocardial function [5]. Additionally, a considerable number of patients are ineligible for or for revascularization, derive limited benefit from PCI and CABG. Consequently, there is an urgent need for new therapeutic approaches in the management of MI.
Increasing experimental evidence reveals that the restoring functional blood and lymphatic vessel networks in the infarcted and peri-infarct areas through angiogenesis and lymphangiogenesis is crucial for MI treatment [6, 7]. Angiogenesis and the establishment of collateral circulation in MI provide oxygen and blood supply to ischemic myocardial tissue, helping tissue repair and enhancing cardiac function [8]. Lymphatic vessels transport interstitial fluid to the lymph nodes and return it to the venous system, thereby potentially reducing cardiac edema [9]. Literature reports indicate that impaired lymphatic vessels post-MI contribute to edema development and worsen cardiac dysfunction [10]. Promoting cardiac lymphangiogenesis can mitigate cardiac edema, inflammation, and fibrosis, thereby enhancing left ventricular function [11, 12]. Members of the vascular endothelial growth factor (VEGF) family like VEGF-A and VEGF-C, play key roles in angiogenesis and lymphangiogenesis [13]. VEGF-A stimulates endothelial cell proliferation via vascular endothelial growth factor receptor 2(VEGFR-2) binding, while VEGF-C promotes lymphangiogenesis by binding to vascular endothelial growth factor receptor 3(VEGFR-3)[14]. Research demonstrates that administering VEGF-C and VEGF-A proteins directly into the heart can foster blood vessel and lymphatic formation, enhance collateral circulation, reduce edema, and restore cardiac function in MI mice model [8, 15]. Thus, targeting angiogenesis and lymphangiogenesis holds promise for therapeutic intervention in MI management.
Ginkgo biloba L., first documented in the ancient Chinese compendium “Ben Cao Gang Mu” [16], uses various parts, including leaves, seeds, and fruits, for medicinal purposes. Shuxuening injection (SXNI), derived from Ginkgo biloba L. leaves extract (GBE), contains key active compounds like ginkgo flavonoid glycosides and ginkgolides [17]. Widely used in treating ischemic cardiovascular conditions like angina pectoris and MI, SXNI has been approved by the National Medical Products Administration of China (Approval No: 14021871) [18]. While SXNI effectively enhances long-term outcomes for patients with MI clinically, its precise pharmacological mechanism remains inadequately elucidated. Recent research indicates that GBE attenuates myocardial ischemic injury by vasodilation and enhancing blood flow in ischemic regions [19]. Bilobalide, another constituent, demonstrates neuroprotective effects through angiogenesis promotion [20]. GBE also facilitates lymphatic circulation, accelerating silica elimination from the lungs by promoting lymphangiogenesis [21]. Here, we demonstrate that SXNI enhances cardiac function and suppresses cardiac fibrosis in MI-induced rats by dual modulation of angiogenesis and lymphangiogenesis.
Macrophages play a crucial role in tissue repair and regeneration following MI [22]. Extensive data indicate that macrophages secrete numerous essential growth factors, which indirectly affect vascular and lymphatic endothelial cells, thereby regulating angiogenesis and lymphangiogenesis [23, 24]. However, whether SXNI can activate macrophages to promote the secretion of such cytokines and subsequently exert indirect pro-angiogenic and pro-lymphangiogenic effects remains unexplored. Therefore, from the perspective of indirect pharmacology, we examined the regulatory effects of SXNI on angiogenic factors within macrophages and assessed its impact on macrophage-mediated functions and pathways in vascular and lymphatic endothelial cells. Furthermore, we investigated the key active components of SXNI that activate macrophages. These investigations aim to elucidate the novel mechanism by which SXNI promotes tissue repair indirectly through immune modulation, providing a new theoretical foundation for its application in MI treatment.
Materials and methods
Reagents
Shuxuening Injection (SXNI, batch number: 22112411) was obtained from Langzhi Wanrong Pharmaceutical Co., Ltd. (Shanxi, China). Each unit of SXNI (10 mL) contains 8.4 mg of total flavonol glycosides and 1.4 mg of ginkgolides. The SXNI manufacturing process was prepared following the standards of the China Food and Drug Administration (YBZ05042017). Ginkgolide B (CHB230911, purity ≥ 98%) and ginkgolide A (CHB230725, purity ≥ 98%) were purchased from Chengdu Chroma- Biotechnology Co., Ltd (Chengdu, China). Quercetin 3-neohesperidoside(JOT-11175, purity ≥ 98%) and rutin (JOT-10229, purity ≥ 98%) was purchased from Chengdu Pufei De Biotech Co., Ltd (Chengdu, China).
Rat MI model and treatment
Male Sprague–Dawley (SD) rats, aged between 6 to 8 weeks and weighing 200 ± 20 g (Beijing HuaFuKang Bioscience Co., Ltd., Beijing, China). All procedures involving experimental animals strictly adhered to the Guidelines for the Care and Use of Experimental Animals and were approved by the Animal Ethics Committee of the First Affiliated Hospital of the Henan University of Chinese Medicine (approval number: YFYDW2023021).
The MI rat model was established by permanent ligation of the left anterior descending coronary artery (LAD) [25]. Rats were anesthetized and intubated using a small rodent ventilator. The LAD coronary artery was ligated with a 6–0 suture to induce MI. The rats in the sham surgical group underwent the same procedure without the ligation of LAD. The rats were randomly divided into 5 groups: (1) sham group, (2) model group, (3) SXNI treatment at a dosage of 1.05 mL/kg/d, (4) SXNI treatment at a dosage of 2.1 mL/kg/d (clinical equivalent dose), (5) SXNI treatment at a dosage of 4.2 mL/kg/d [26], and (6) and Captopril treatment at a dosage of 4.5 mg/kg/d [26, 27], Starting from the first day after MI induction, SXNI was administered through intraperitoneal (i.p.) injection once daily for four weeks. Captopril was suspended in saline and administered by gavage for four weeks. The sham and model groups received equivalent amounts of normal saline via i.p. injection once daily for the same duration.
Echocardiography
Four weeks after MI, cardiac function was assessed using echocardiography with a Vevo 1100 ultrasound system (Visual Sonic, Canada). Two-dimensional M-mode images of the left ventricular short-axis section and long-axis sections were obtained. The left ventricular ejection fraction (LVEF), left ventricular fractional shortening (LVFS), systolic left ventricular internal dimension (LVID s), and diastolic left ventricular internal dimension (LVID d) were calculated using Vevo 1100 software.
Histologic analysis
For H&E staining, tissue slices were stained with H&E and the histopathologic damage was assessed using a light microscope (Nikon, Tokyo, Japan). Masson staining was conducted based on the instructions provided by the manufacturer to observe the cardiac fibrosis. In the stained sections, the red areas indicated healthy muscle fibers, while the blue areas represented fibrosis with collagen deposition.
Immunofluorescence (IF) analysis and Immunohistochemistry (IHC)
Myocardial cell apoptosis was assessed through TUNEL staining, with apoptosis ratios quantified using ImageJ software. IF and IHC staining procedures were conducted as previously described [28]. The following antibodies were used: monoclonal anti-CD31 antibody (1:200, AF6191, Affinity, Jiangsu, China) for staining microvessels, polyclonal anti-VEGF-A antibody (1:200, SC-7269, Santa Cruz Biotechnology, USA) for staining VEGF-A protein, monoclonal anti-LYVE-1 antibody (1:200, #67,538, CST, Boston, USA) for staining lymphatic vessels, and monoclonal anti-VEGF-C antibody (1:200, SC-374628, Santa Cruz Biotechnology, USA) for staining VEGF-C protein. Images of the heart tissue sections were analyzed using ImageJ software.
Gravimetry
After four weeks of administration, heart tissues were harvested and dried at 105℃ for 12 h. The heart water content was determined using the wet weight versus dry weight method. The formula used to calculate the heart water content is as follows:
\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\text{Heart water content }(\mathrm{\%})=(\text{wet heart weight}-\text{dry heart weight})/\text{wet heart weight }\times 100\mathrm{\%}$$\end{document}ELISA
To assess serum biomarkers related to myocardial injury, inflammation, angiogenesis, and lymphangiogenesis in MI rats, serum levels of brain natriuretic peptide (BNP) (E-EL-R0126c), tumor necrosis factor-α (TNF-α) (E-EL-R2856c), interleukin-1β (IL-1β) (E-EL-R0012), interleukin-6 (IL-6) (E-EL-0015c), VEGF-A (E-EL-R2603c), and VEGF-C (E-EL-R1057c) were quantified using ELISA kits. To examine the impact of SXNI and the active ingredients on VEGF-A and VEGF-C proteins secretion in macrophages, Raw264.7 cells were treated with SXNI (10μL/mL, 25μL/mL, 50μL/mL), ginkgolide A(40μM), ginkgolide B(40μM), rutin(40μM) or quercetin 3-neohesperidoside(40μM) for 24 h, followed by a collection of supernatants. The levels of VEGF-A (E-MSEL-M0005) and VEGF-C (E-EL-M1230c) proteins in the supernatants were measured using ELISA kits from Elabscience Biotechnology (Wuhan, China).
Cell culture
The macrophage cell line Raw264.7 and the vascular endothelial cell line human umbilical vein endothelial cells (HUVEC) were obtained from the cell bank of the Chinese Academy of Sciences (Shanghai, China). The lymphatic endothelial cell line SV40-transformed mouse endothelial cells (SVEC4-10) was acquired from Pricella Biotechnology Co., Ltd. (Wuhan, China). All cell lines were cultured in DMEM (Gibco, NY, USA) supplemented with 10% FBS.
Cell viability
The cytotoxicity of SXNI was assessed by treating cells with SXNI at concentrations ranging from 5 to 200 µL/mL for a duration of 24 h. Cell viability of HUVECs and SVEC4-10 cells influenced by macrophages was assessed using CCK-8 assays. Specifically, HUVECs or SVEC4-10 cells were cultured in medium containing 50 μL/mL SXNI, or 50 μL/mL SXNI-treated Raw264.7 cell supernatants, or non-SXNI-treated Raw264.7 cell supernatants for 24 h. Raw264.7 cell supernatants were collected after 24 h of incubation with 50 μL/mL SXNI or without SXNI. Similarly, Cell viability after treatment with ginkgolide A, ginkgolide B, rutin or quercetin 3-neohesperidoside was evaluated using a CCK-8 assay. Raw264.7 cells were treated with ginkgolide A, ginkgolide B, rutin or quercetin 3-neohesperidoside at concentrations ranging from 2.5 to 320 μM for 24 h. Subsequently, 100 µL of CCK-8 (10 µg/mL) was added. The formula for calculating the cell viability rate was as follows:
\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\text{Cell viability }(\mathrm{\%}) = (\mathrm{Atest}-\mathrm{Ablank})/(\mathrm{Acontrol}-\mathrm{Ablank}) \times 100\mathrm{\%}$$\end{document}qRT-PCR
Total RNA from heart tissue or Raw264.7 cells was extracted by using the RNA-Quick Purification Kit following four weeks of SXNI administration in rats or treatment of Raw264.7 cells with SXNI (10μL/mL, 25μL/mL, 50μL/mL), ginkgolide A (20, 40, 80 μM), ginkgolide B(20, 40, 80 μM), rutin(20, 40, 80 μM) or quercetin 3-neohesperidoside(20, 40, 80 μM) for 3 h (Vegf-a) and 9 h (Vegf-c). cDNA was synthesized using the Fast ALL-in-One RT Kit. qRT-PCR was conducted on a Cobas z480 instrument (Roche, Basel, Switzerland) with β-actin used as an internal reference gene for normalization. Primer sequences are shown in Supplementary Table S1.
Migration assay
Wounds were established using a 200 μL pipette tip. Cells were then cultured in a medium containing 50 μL/mL SXNI, or 50 μL/mL SXNI-treated Raw264.7 cell supernatants, or non-SXNI-treated Raw264.7 cell supernatants for 12 h. Raw264.7 cell supernatants were collected after 24 h of incubation with 50 μL/mL SXNI or without SXNI. Photographs were taken at 0 and 12 h after scratching using a microscope (Leica, Wetzlar, Germany). Migration rates of HUVECs and SVEC4-10 cells were quantified using ImageJ software.
Tube formation assay
The 96-well plates were coated with Matrigel matrix and then polymerized at 37 °C for 1 h. After the Matrigel matrix solidified, HUVECs and SVEC4-10 cells at a concentration of 1.25 × 10^5^ cells/mL were suspended in 50 μL/mL SXNI, or 50 μL/mL SXNI-treated Raw264.7 cell supernatants, or non-SXNI-treated Raw264.7 cell supernatants. The cells were then added onto the surface of the Matrigel matrix and then incubated for 3 h. The vascular structures formed were conceptualized and photographed with a microscope.
Western blot
Heart tissue and cell lysates were extracted using high-efficiency RIPA rapid lysis buffer. Western blot analysis following established procedures [29]. Antibody information is shown in Supplementary Table S2. Images were captured using the CLiN chemiluminescence imaging system 6000 (Shanghai, China). HUVECs and SVEC4-10 cells were incubated in a medium containing 50 μL/mL SXNI, or 50 μL/mL SXNI-treated Raw264.7 cell supernatants, or non-SXNI-treated Raw264.7 cell supernatants, or 50 ng/mL VEGF-A protein, or 50 ng/mL VEGF-C protein for 30 min.
UPLC-QE-MS analysis of SXNI
A 100 μL SXNI sample was mixed with 900 μL of methanol in a 1.5 mL centrifuge tube. Ultrasonic extraction in ice water bath for 60 min, centrifugation for 10 min (12,000 rpm, 4 °C), and 200 μL supernatant was taken for analysis. Separation was performed on an ACQUITY UPLC HSS T3 column (2.1 × 100 mm, 1.8 µm) maintained at 45°C, with a flow rate of 0.35 mL/min and an injection volume of 2 µL. The mobile phase consisted of 0.1% formic acid in water (A) and acetonitrile (B), with gradient elution conditions outlined in Supplementary Table S3. Ion source: HESI; sample mass spectrometry signal acquisition was performed in positive and negative ion scanning modes, respectively. The mass spectrum parameters: the spray voltage of positive and negative ions is divided into 3800V and − 3200V. The capillary temperature is 320°C, and the auxiliary gas heater temperature is 250°C. The full-scan data acquisition range was set from 100 to 1500 m/z.
Molecular docking
Molecular docking was employed to investigate the interactions between the components of the SXNI and the VEGF-A and VEGF-C proteins. The 3D structures of the compounds were downloaded from PubChem, while the 3D structures of the VEGF-A and VEGF-C proteins were obtained from the PDB database (https://www.rcsb.org/). The docking and scoring between the proteins and the compounds were conducted using the CB-Codk2 website (https://cadd.labshare.cn/cb-dock2/index.php). The 2D visualisation was conducted using Discovery Studio software.
Statistical analysis
All experimental data were analyzed using GraphPad Prism 8.0 software. The results are presented as the mean ± standard deviation. For data following a normal distribution, one-way analysis of variance (ANOVA) was applied to compare differences among multiple groups. In cases of homogeneous variances, the least significant difference (LSD) post hoc test was used; for heterogeneous variances, the Games-Howell test was employed. For data not conforming to a normal distribution, the non-parametric Kruskal–Wallis test was utilized. P < 0.05 was considered statistically significant.
Results
SXNI inhibited ventricular remodeling and protected heart function in MI rats
To assess the cardioprotective effects of SXNI, we induced an MI rat model. After four weeks of SXNI treatment, cardiac function was evaluated by echocardiography. The model group exhibited significantly reduced LVEF and LVFS, along with increased LVID s and LVID d left ventricular internal dimensions (^##^P < 0.01, ^###^P < 0.001), rats treated with SXNI (2.1 mL/kg and 4.2 mL/kg) and captopril revealed higher LVEF and LVFS values compared to the model group, while LVID d and LVID s were lower (^^P < 0.05, ^**^P < 0.01, Fig. 1A–E), indicating the protective effect of SXNI on cardiac function. Moreover, the HW to BW ratio, indicative of cardiac hypertrophy, was elevated in the model group (^###^P < 0.001). Treatment with SXNI (2.1 and 4.2 mL/kg) and captopril significantly reversed this increase (^^P < 0.05, ^^P < 0.001, Fig. 1F). Since the efficacy of SXNI (2.1 and 4.2 mL/kg) was comparable to that of captopril, which confirmed its protective effects in the MI model, these two doses were selected for subsequent experiments.Fig. 1SXNI suppressed ventricular remodeling and protected heart function in MI rat. A M-mode echocardiographic images. B–E Quantification results of LVEF, LVFS, LVID d and LVID s values at four weeks after MI in each group (n = 8). F Quantification of HW/BW (n = 8). G Images of H&E staining. H Images of Masson trichrome staining. I Quantification of fibrosis areas (n = 3). Data are expressed as mean ± SD. ^##^P < 0.01, ^###^P < 0.001, vs the sham group; P < 0.05, **P < 0.01, ***P < 0.001, vs the model group; ns, no significant difference
H&E staining was used to assess the pathological changes in cardiac tissue post-MI. In the model group, the structure of myofibrils was notably disordered and irregularly arranged. Cardiomyocytes in the infarct border zone exhibited edema, uneven cytoplasm, and irregular nuclear changes. In contrast, these histopathologic abnormalities were alleviated, and cell morphology was enhanced in rats treated with SXNI (2.1 and 4.2 mL/kg) and captopril (Fig. 1G). Masson staining demonstrated that the collagen deposition area was significantly increased in the model group compared to the sham group (^###^P < 0.001). However, SXNI (2.1 and 4.2 mL/kg) and captopril administration reduced the collagen deposition area (^^P < 0.001, Fig. 1H, I). Additionally, the treatment with SXNI can significantly reduce the area of myocardial infarction in rats (^^P < 0.01, ^^P < 0.001, Fig. S1A). To further examine the effects of SXNI on myocardial injury, BNP serum levels were measured. Treatment with SXNI (2.1 and 4.2 mL/kg) resulted in a reduction of BNP levels (^^P < 0.05, *^***^P < 0.001, Fig. S1B). The above results indicate that SXNI inhibited ventricular remodeling and protected cardiac function in MI rats.
SXNI inhibits myocardial apoptosis after MI in rats
Myocardial apoptosis is a significant contributor to ventricular remodeling and heart failure following MI [30]. Preventing myocardial apoptosis is crucial for enhancing cardiac function [31]. To assess whether SXNI mitigates MI injury by inhibiting myocardial apoptosis, TUNEL staining was conducted. A substantial number of apoptotic cells were observed in the MI border area of the model group (^###^P < 0.001), whereas the apoptotic cells were reduced in the SXNI treatment groups (2.1 and 4.2 mL/kg) (^^P < 0.01, Fig. 2A, B). Additionally, qRT-PCR analysis of heart tissue revealed that the mRNA expression levels of Bax were significantly decreased (^^P < 0.001) in the SXNI treatment groups (2.1 and 4.2 mL/kg), while the mRNA expression levels of Bcl-2 were significantly increased (^^P < 0.01; ^^P < 0.001) (Fig. 2C, D). The protein expression levels of BAX (^#^P < 0.05) and cleaved-caspase-3 (^##^P < 0.01) were elevated in the model group. However, SXNI treatment (2.1 and 4.2 mL/kg) resulted in a reduction of BAX (^^P < 0.01) and cleaved-caspase-3 (^^P < 0.05) protein expression levels, and an increase in BCL-2 (^^P < 0.05) protein expression levels (Fig. 2E).Fig. 2SXNI inhibits myocardial apoptosis after MI. A Images of TUNEL immunofluorescence. B Intensity of apoptotic cells after four weeks of MI model (n = 3). C, D qRT-PCR indicating relative mRNA levels of Bax and Bcl-2 in tissue (n = 6). E Apoptosis-related proteins expression in tissue measured by Western blot (n = 3). Data are expressed as mean ± SD. ^#^P < 0.05*,* ^##^P < 0.01*, ^###^P < 0.001, vs the sham group; ^^P < 0.05, ^****^P < 0.01, ^*****^P < 0.001, vs the model group; ns, no significant difference
SXNI promotes angiogenesis and lymphangiogenesis thereby alleviating cardiac edema and inflammation after MI
MI-induced cardiac injury leads to edema and inflammation, which contribute to cardiac dysfunction. To assess the protective effects of SXNI, we first evaluated its impact on cardiac edema and inflammation. Cardiac water content was significantly higher in the model group than in the sham group (^###^P < 0.001). Both doses of SXNI (2.1 and 4.2 mL/kg) dramatically reduced this elevation (^^P < 0.01; ^^P < 0.001; Fig. 3A). Concurrently, SXNI treatment significantly lowered serum levels of the pro-inflammatory factors IL-1β, TNF-α, and IL-6 compared to the model group (^^P < 0.05, ^^P < 0.01; ^^P < 0.001, Fig. 3B–D).Fig. 3SXNI induces angiogenesis and lymphangiogenesis, thereby reducing cardiac edema and inflammatory response caused by MI. A Cardiac water content (%) in rats is measured (n = 7). B–D ELISA analysis of serum IL-1β, TNF-α, and IL-6 levels in rats treated with SXNI at four weeks post-MI is conducted (n = 8). E Images of CD31 staining are presented and Quantification of the CD31^+^ area was conducted (n = 3). F Images of LYVE-1 staining are depicted and quantification of the LYVE-1^+^ area is conducted (n = 3). Data are expressed as mean ± SD. ^##^P < 0.01, ^###^P < 0.001, vs the sham group; ^^P < 0.05, ^****^P < 0.01, ^***^P < 0.001, vs the model group; ns, no significant difference
Since angiogenesis establishes compensatory collateral circulation to alleviate myocardial ischemia and improves the local inflammatory microenvironment [32], we examined whether SXNI promotes this process. As depicted in Fig. 3E, the mean intensities of CD31 in the SXNI treatment groups (2.1 and 4.2 mL/kg) was significantly increased (^^P < 0.01). Further tests revealed that SXNI (4.2 mL/kg) reduced cardiac vascular leakage and restored damaged cardiac tissue blood vessels in MI rats (^^P < 0.001, Fig. S1C). We next evaluated whether SXNI also influences cardiac lymphangiogenesis, which promotes the resolution of inflammation and edema after MI [33]. Immunohistochemical staining for LYVE-1 revealed a substantially higher mean density in the SXNI-treated groups compared to the model group (^**^P < 0.001, Fig. 3F). These results suggest that SXNI alleviates cardiac edema and inflammation following MI, potentially through the dual promotion of angiogenesis and lymphangiogenesis.
SXNI promotes angiogenesis and lymphangiogenesis via regulating VEGF-A/VEGFR-2 and VEGF-C/VEGFR3 pathways
VEGF-A, a key regulator of angiogenesis, was investigated. VEGF-A protein levels in both heart tissues and serum from rats treated with SXNI (2.1 and 4.2 mL/kg) were significantly higher than those in the model group (^^P < 0.01, Fig. 4A, B). Given that VEGFR-2 stands out as the primary receptor for VEGF-A mediating angiogenic activity [34]. WB was used to assess whether SXNI promotes angiogenesis in MI by regulating the VEGF-A/VEGFR-2 pathway. As depicted in Fig. 4C, SXNI treatment groups (2.1 and 4.2 mL/kg) significantly promoted VEGF-A (^*^P < 0.05, ^^P < 0.01) and p-VEGFR2 (^^P < 0.001, ^^P < 0.01) compared with the model group.Fig. 4SXNI promotes angiogenesis and lymphangiogenesis via regulating VEGF-A/VEGFR-2 and VEGF-C/VEGFR3 pathways. A Images of VEGF-A staining are depicted and IHC quantification of VEGF-A was conducted (n = 3). B ELISA analysis was used to determine serum VEGF-A levels in rats treated with SXNI at four weeks post-MI (n = 8). C Relative protein expression levels of VEGF-A and p-VEGFR2 in cardiac tissue were assessed (n = 3). D Images of VEGF-C staining are depicted and IHC quantification of VEGF-C is conducted (n = 3). E ELISA analysis of serum VEGF-C levels in rats treated with SXNI at four weeks post-MI is conducted (n = 8). F Relative VEGF-C and VEGFR3 protein expression levels in cardiac tissue are assessed (n = 3). Data are expressed as mean ± SD. ^#^P < 0.05,* ^##^P < 0.01*, ^###^P < 0.001, vs the sham group; ^^P < 0.05, ^****^P < 0.01, ^***^P < 0.001, vs the model group; ns, no significant difference
VEGF-C, a key regulator of lymphangiogenesis, was assessed. The regulatory effect of SXNI on cardiac lymphangiogenesis was further confirmed by IHC staining for VEGF-C. SXNI treatment (2.1 and 4.2 mL/kg) significantly increased VEGF-C protein levels in both cardiac tissues (^^P < 0.05) and serum (^^P < 0.01, ^^P < 0.001) compared to the model group (Fig. 4D, E). Given that activation of the VEGF-C/VEGFR3 axis selectively stimulates cardiac lymphangiogenesis, the effect of SXNI on the VEGF-C/VEGFR3 signaling pathway in MI was further studied. WB indicated that SXNI treatment groups (2.1 and 4.2 mL/kg) significantly promoted VEGF-C (^^P < 0.05) and VEGFR3 (^^P < 0.05) when compared with the model group (Fig. 4F).
SXNI has no direct affection to promoting angiogenesis and lymphangiogenesis in vitro
The effect of SXNI on the viability of HUVECs was assessed, and it was discovered that SXNI exhibited no toxicity in HUVECs at concentrations of 5–50 μL/mL (Fig. 5A). Therefore, we chose the maximum non-toxic dose (50 μL/mL) to conduct subsequent studies. Notably, the proliferative capacity of HUVECs remained unaffected when directly incubated with 50 μL/mL SXNI (SXNI-50) (Fig. 5B). Moreover, we found that SXNI-50 did not directly enhance endothelial cell migration (Fig. 5C). The effect of SXNI on angiogenesis was elucidated by analyzing the formation of capillary-like tubes in a Matrigel matrix. The SXNI-50 group did not significantly enhance tube formation in endothelial cells (Fig. 5D). Furthermore, we saw that SXNI exhibited no toxicity in SVEC4-10 cells at concentrations of 5–50 μL/mL (Fig. 5E). Then, 50 μL/mL of SXNI was selected for subsequent studies. Similarly, we also found that the SXNI-50 group could not directly promote SVEC4-10 cell proliferation, migration, and tubule formation (Fig. 5F–H). Overall, these results indicate that SXNI does not directly act on vascular or lymphatic endothelial cells to enhance their key biological activities, suggesting that its pro-angiogenic and pro-lymphangiogenic effects observed in vivo may be mediated through indirect mechanisms (Fig. 5I).Fig. 5SXNI does not directly promote the biological activity of HUVECs and SVEC4-10 cells. A Cell viability of HUVECs cells after SXNI treatment (n = 6). B The effect of SXNI on the proliferation of HUVECs cells (n = 6). C The effect of SXNI on the migration of HUVECs cells (n = 4). D The effect of SXNI on tubule formation of HUVECs cells (n = 4). E Cell viability of SVEC4-10 cells after SXNI treatment (n = 6). F The effect of SXNI on the proliferation of SVEC4-10 cells (n = 6). G The effect of SXNI on the migration of SVEC4-10 cells (n = 4). H The effect of SXNI on tubule formation of SVEC4-10 cells (n = 3). I Schematic diagram showing the SXNI has no direct effect on endothelial cells. ns, no significant difference
SXNI promotes VEGF-A and VEGF-C proteins release in Raw264.7 cells
Macrophages are the primary source of the pro-angiogenesis and pro-lymphangiogenesis factors, including VEGF-A and VEGF-C, and play a key role in the cardiac repair process post-MI [35, 36]. Given that SXNI did not directly enhance the biological functions of vascular or lymphatic endothelial cells, we hypothesized that its in vivo pro-angiogenic and pro-lymphangiogenic effects might be mediated indirectly through immune cells. Macrophages, in particular, are crucial for regulating angiogenesis and lymphangiogenesis and facilitating tissue repair after MI. Therefore, we next investigated whether SXNI could exert its effects by modulating cytokine secretion from macrophages, aligning with an indirect pharmacology perspective. To examine this, the cytokines secreted by Raw264.7 cells were analyzed. Initially, the effect of SXNI on the viability of Raw264.7 cells was assessed, revealing no toxicity at concentrations below 100 μL/mL (Fig. 6A). Next, the regulatory effects of different concentrations of SXNI on Vegf-a and Vegf-c mRNA levels in Raw264.7 cells were examined. As depicted in Fig. 6B, C, SXNI dose-dependently increased the mRNA levels of Vegf-a (^^P < 0.05, ^^P < 0.01) and Vegf-c (^^P < 0.01) in Raw264.7 cells. Additionally, the effects of SXNI on the secretion of VEGF-A and VEGF-C proteins in Raw264.7 cells were examined by ELISA. Compared to the control group, SXNI increased the levels of VEGF-A depending on the dosage (^^P < 0.01, ^^P < 0.001) and VEGF-C (^^P < 0.05, ^**^P < 0.01) in the supernatants of Raw264.7 cells (Fig. 6D, E). In conclusion, SXNI activates macrophages and promotes the release of VEGF-A and VEGF-C by macrophages.Fig. 6SXNI promotes the protein release of angiogenesis factors and lymphangiogenesis factors in Raw264.7 cells. A Cell viability of Raw264.7 cells after SXNI treatment (n = 6). B, C Relative Vegf-a and Vegf-c mRNA levels in Raw264.7 cells after following 3 h or 9 h of SXNI exposure (n = 3 or 4). D, E VEGF-A and VEGF-C proteins released by Raw264.7 cells after being treated with SXNI for 24 h (n = 3 or 4). Data are expressed as mean ± SD. ^^P < 0.05, ^^P < 0.01*, *^***^P < 0.001, vs the ctrl group; ns, no significant difference
SXNI promotes angiogenesis and lymphangiogenesis in vitro by activating Raw264.7 cells
To further explore the indirect pharmacological mechanism by which SXNI enhances vascular and lymphatic regeneration through macrophage activation, we conducted a series of conditioned medium-based bioactivity assays, as illustrated in the experimental schematics depicted in Fig. 7A, E. First, to examine the indirect pro-angiogenic role of SXNI, HUVECs were incubated with supernatants from Raw264.7 cells treated with 50 μL/mL SXNI (s-SXNI-50) or vehicle control (s-Ctrl). The s-SXNI-50 supernatant significantly promoted the proliferation, (^^P < 0.001, Fig. 7B) migration (^^P < 0.001, Fig. 7C), and tube formation (^^P < 0.05, Fig. 7D). These results demonstrate that SXNI does not act on HUVECs directly but rather stimulates macrophages to release soluble factors that subsequently enhance key angiogenic activities of vascular endothelial cells.Fig. 7SXNI promotes angiogenesis and lymphangiogenesis in vitro by activating Raw264.7 cells. A Schematic diagram showing the indirect influence of SXNI on endothelial cells through macrophages. B Cell viability of HUVECs when incubated with supernatants of SXNI-treated macrophages (n = 6). C SXNI-treated macrophage supernatants on HUVEC migration (n = 4). D SXNI-treated macrophage supernatants on HUVEC tube formation (n = 4). E Schematic diagram showing the indirect influence of SXNI on lymphatic endothelial cells through macrophages. F Cell viability of SVEC4-10 when incubated with supernatants of SXNI-treated macrophages (n = 6). G SXNI-treated macrophage supernatants on SVEC4-10 migration (n = 4). H SXNI-treated macrophage supernatants on SVEC4-10 tube formation (n = 3). Data are expressed as mean ± SD. ^^P < 0.05, ^^P < 0.01, ^***^P < 0.001, vs the s-ctrl group; ns, no significant difference
Similarly, we investigated the indirect pro-lymphangiogenic effect mediated by macrophages. When SVEC4-10 lymphatic endothelial cells were incubated with the s-SXNI-50 supernatant, their proliferation (^^P < 0.001, Fig. 7F), migration (^^P < 0.001, Fig. 7G), and tubule formation (^**^P < 0.01, ^*^P < 0.05, Fig. 7H) were all significantly enhanced relative to the s-Ctrl group. Overall, these conditioned medium experiments provide direct in vitro evidence supporting an indirect pharmacology mechanism. SXNI activates macrophages to secrete bioactive signals, which in turn promote both angiogenesis and lymphangiogenesis by enhancing the biological activities of their respective endothelial cells.
The AKT and ERK signaling pathways are activated by SXNI through Raw264.7 cells
Since VEGFR-2 stands out as the primary receptor mediating the biological effects of VEGF-A, and the downstream signaling pathway mediated by VEGFR-2, including PI3K/AKT and ERK, plays a key role in angiogenesis [34], we used western blot analysis to assess the levels of p-VEGFR2, p-AKT, and p-ERK in HUVECs after treatment with the supernatant of SXNI-treated Raw264.7 cells. As depicted in Fig. 8A, compared with the Ctrl group, the protein levels of p-VEGFR2 were significantly increased in the s-Ctrl (^^P < 0.001), s-SXNI-50 (^^P < 0.001), and VEGF-A (^^P < 0.01) groups, with the highest levels observed in the s-SXNI-50 group. Similarly, the protein levels of p-AKT and p-ERK were significantly increased in the s-SXNI-50 groups (^^P < 0.001, ^**^P < 0.01).Fig. 8. The AKT and ERK signaling pathways are activated by SXNI through Raw264.7 cells. A Quantitative analysis of p-VEGFR2, p-AKT and p-ERK protein expression levels in HUVECs after 30 min incubation with the supernatant of Raw264.7 cells treated with 50 μL/mL SXNI for 24 h (n = 3). B Western blot and quantitative analysis of VEGFR3, p-AKT, and p-ERK protein expression levels in SVEC4-10 cells after 30 min of incubation with the supernatant of Raw264.7 cells treated with 50 μL/mL SXNI for 24 h (n = 3). C The relative p-AKT and p-ERK protein expression levels in cardiac tissue (n = 3). Data are expressed as mean ± SD. ^^P < 0.05, ^^P < 0.01, ^**^P < 0.001, vs the ctrl group; ^#^P < 0.05, ^##^P < 0.01, ^###*^P < 0.001, vs the s-ctrl group; ^&^P < 0.05, ^&&^P < 0.01, versus the model group; ns, no significant difference
As for lymphangiogenesis, we further assess the levels of VEGFR3, p-AKT and p-ERK in SVEC4-10 cells after treatment with the supernatant of SXNI-treated Raw264.7 cells. As depicted in Fig. 8B, compared with the Ctrl group, the protein levels of VEGFR3 were dramatically increased in the s-Ctrl (^^P < 0.05), s-SXNI-50 (^**^P < 0.01), and VEGF-C (^^P < 0.05) groups, with the highest levels observed in the s-SXNI-50 group. Similarly, the protein levels of p-AKT and p-ERK were significantly increased in the s-SXNI-50 groups (^**^P < 0.001, ^^P < 0.05). As depicted in Fig. 8C, SXNI (2.1 mL/kg and 4.2 mL/kg) treatment groups significantly promoted p-AKT (^&^P < 0.05, ^&&^P < 0.01) and p-ERK (^&^P < 0.05) when compared with the model group. Overall, these findings elucidate the key steps in the indirect pharmacological mechanism of SXNI: it activates macrophages to secrete VEGFs, such as VEGF-A and VEGF-C, which act on vascular endothelial cells and lymphatic endothelial cells, triggering the VEGFR2 and VEGFR3-mediated AKT and ERK signaling pathways, thereby promoting angiogenesis and lymphangiogenesis.
Identification of compounds of SXNI using UPLC-QE-MS
The identification of the compounds was based on accurate mass number, secondary fragment and isotope distribution, and the TCM database was used for qualitative analysis. The SXNI sample was analyzed in both positive and negative ionization modes, with base peak ion chromatograms (BPC) shown in Fig. 9A, B. According to the multi-stage mass spectrometry information of the sample and the standard database, Seventy compounds were identified from SXNI, and their molecular formulas, retention times, measured mass, and ion characteristics are detailed in Table 1.Fig. 9. Identification of main compounds of SXNI using UPLC-QE-MS. BPC of the SXNI in positive ion mode (A) and negative ion mode (B) using UPLC-QE-MSTable 1Results of the identification of the main components of SXNI samplesNo.IdentificationFormulaRT (min)Theoretical (m/z)Measured (m/z)Ion modeError (ppm)Peak areaFragment ions1Scyllo-InositolC_6_H_12_O_6_0.795179.0557179.05591[M − H]^−^1.12697,968,769.3101.02448; 113.02454; 119.034912D-ononitolC_7_H_14_O_6_0.806217.0679217.06809[M + Na]^+^0.9240,523,661.79217.06831; 217.10362; 218.099643Quinic acidC_7_H_12_O_6_0.845191.0557191.05595[M − H]^−^1.05663,046,449.2191.0558541-O-alpha-D-Glucopyranosyl-D-mannitolC_12_H_24_O_11_0.867343.1232343.12392[M − H]^−^2.04100,258,521.5119.03495, 297.11899, 343.123845Shikimic acidC_7_H_10_O_5_0.914173.0453173.04545[M − H]^−^0.58419,805,429.5142.9983; 143.0347; 155.03496ArbutinC_12_H_16_O_7_1.44271.0819271.08208[M − H]^−^0.741,330,804.743190.4591; 224.8161; 287.85017GastrodinC_13_H_18_O_7_1.826331.1025331.10304[M + FA − H]^−^1.5197,400,323.09285.09753; 305.17075; 331.103068Vanillic acid 4-beta-D-glucosideC_14_H_18_O_9_3.25329.087329.0874[M − H]^−^1.229,053,418.087205.8613; 261.8507; 329.08739Protocatechuic acidC_7_H_6_O_4_3.459153.0191153.01918[M − H]^−^0.6515,683,106.27109.02934104'-O-beta-D-Glucosyl-cis-p-coumarateC_15_H_18_O_8_3.857371.0974371.09793[M + FA − H]^−^1.357,093,225.323163.04013; 235.92625; 325.0925911AndrosinC_15_H_20_O_8_4.043327.1078327.10819[M − H]^−^1.227,575,000.103165.0553; 190.92842; 327.1071512SyringinC_17_H_24_O_9_4.163395.1299395.13056[M + Na]^+^1.778,670,467.34395.1311613ClemastaninBC_32_H_44_O_16_4.232729.2627729.26192[M + FA − H]^−^− 1.123,567,169.21521.20209; 522.20538; 683.2556814Pinoresinol diglucosideC_32_H_42_O_1_64.418727.2471727.24632[M + FA − H]^−^− 1.13,145,501.713358.13687; 519.18671; 681.2404215Kaempferol 3,7-diglucosideC_27_H_30_O_16_4.433655.1546655.15308[M + FA − H]^−^− 2.2926,956,834.2610.1445; 655.1509; 656.149016ManghaslinC_33_H_40_O_20_4.473757.219757.21879[M + H]^+^− 0.263,642,757.127303.04984; 465.1033; 611.1617417Quercetin 3-sophorosideC_27_H_30_O_17_4.539649.1373649.13741[M + Na]^+^0.1514,470,918.99331.09949; 649.1380618Myricetin 3-rutinosideC_27_H_30_O_17_4.541627.1554627.15552[M + H]^+^0.1625,331,233.4585.02906; 319.04462; 481.0977219RoseosideC_19_H_30_O_8_4.597409.1825409.18289[M + Na]^+^0.9824,102,380.18229.18001; 409.11597; 409.1834120MauritianinC_33_H_40_O_19_4.618741.2244741.22396[M + H]^+^− 0.5415,009,091.32287.05493; 449.10794; 595.1664421Myricetin 3-galactosideC_21_H_20_O_13_4.636479.0821479.08261[M − H]^−^1.045,724,091.903415.1024; 329.065622Myricetin 3-glucosideC_21_H_20_O_13_4.636481.0969481.09726[M + H]^+^0.8312,251,495.33401.1242; 371.113623TyphaneosideC_3_4H_42_O_20_4.641771.2346771.2344[M + H]^+^− 0.2636,354,222.04317.06531; 479.1189; 625.1768224Megastigm-7-ene-3,5,6,9-tetraolC_13_H_24_O_4_4.659227.1632227.16403[M + H-H_2_O]^+^3.527,280,035.257191.1430; 209.1535; 227.163825Secoisolariciresinol monoglucosideC_26_H_36_O_11_4.685523.2181523.21832[M − H]^−^0.385,187,610.83273.0758; 257.080826AilanthoneC_20_H_24_O_7_4.69399.1406399.14101[M + Na]^+^110,478,066.7399.1413; 399.1967827Quercetin 3-neohesperidosideC_27_H_30_O_16_4.745611.1606611.16061[M + H]^+^0109,291,492.285.02905; 303.04977; 465.1031828RutinC_27_H_30_O_16_4.746609.1457609.14594[M − H]^−^0.33267,436,657.6301.03436; 609.14557; 610.1491729Quercetin 3-(6''-malonylglucoside)C_24_H_22_O_15_4.881531.0733531.07536[M − H_2_O − H]^−^3.958,478,318.587131.0491; 121.0284308-Hydroxyluteolin 7-glucosideC_21_H_20_O_12_4.882465.1018465.10226[M + H]^+^1.0843,157,660.84133.1012; 119.085531Laricitrin 3-glucosideC_22_H_22_O_13_4.885493.0982493.09849[M-H]^−^0.6150,721,078.01247.0601; 269.044432Quercetin 3-glucosyl-(1- > 2)-rhamnosideC_27_H_30_O_16_4.885611.16611.16033[M + H]^+^0.4912,120,895.59345.06046; 449.10837; 611.1600333IsoquercitrinC_21_H_20_O_12_4.887463.0876463.08795[M − H]^−^0.6576,858,409.84301.03442; 463.08749; 464.0903634RobinetinC_15_H_10_O_7_4.897303.0489303.04944[M + H]^+^1.6552,371,447.72303.04974, 304.0528335NicotiflorinC_27_H_30_O_15_4.935593.1514593.15132[M − H]^−^− 0.17182,310,653.8285.03989; 593.15088; 594.1538136Kaempferol 3-robinobiosideC_27_H_30_O_15_4.938595.1654595.16557[M + H]^+^0.3485,709,146.1685.02903; 287.05496; 449.10797376-Methoxyluteolin 7-glucosideC_22_H_22_O_12_4.962479.1174479.11789[M + H]^+^1.045,149,500.22153.018238Pyrocatechuic acidC_7_H_6_O_4_4.968153.0191153.0192[M − H]^−^0.654,157,542.777109.02944; 135.0086439NarcissinC_28_H_32_O_16_4.973623.1617623.16171[M − H]^−^0169,395,453.3315.05078; 623.16193; 624.1652240Syringetin 3-rutinosideC_29_H_34_O_17_5.005655.1867655.18684[M + H]^+^0.1516,588,227.8985.02903; 347.07608; 509.129341Episyringaresinol 4'-O-beta-D-glncopyranosideC_28_H_36_O_13_5.081579.2083579.20832[M − H]^−^09,017,999.923325.1071; 311.127842Kaempferol 3-glucosyl-(1- > 2)-rhamnosideC_27_H_30_O_15_5.094595.1654595.16558[M + H]^+^0.3468,530,973.15397.09177; 433.11313; 595.1609543LuteolosideC_21_H_20_O_11_5.136447.0927447.09297[M − H]^−^0.6785,925,363.02300.02701; 447.09256; 448.0953144AstragalinC_21_H_20_O_11_5.146449.1066449.10722[M + H]^+^1.3454,045,169.64315.0499; 207.065245Genistein 7-O-glucosideC_21_H_20_O_10_5.155433.1119433.11244[M + H]^+^1.1528,601,566.27271.06; 433.1127646Isorhamnetin 3-glucosideC_22_H_22_O_12_5.16477.1027477.10335[M − H]^−^1.2622,113,306.17315.05029; 477.10303; 478.1067547Ginkgolide JC_20_H_24_O_10_5.184469.1343469.13467[M + FA − H]^−^0.85201,913,615.7367.13882; 423.12912; 469.1345248Ginkgolide CC_20_H_24_O_11_5.223879.2575879.25704[2M − H]^−^− 0.57211,176,548.6383.1344; 411.1294; 439.124349Luteolin 3'-methyl ether 7-glucosideC_22_H_22_O_11_5.227461.1075461.10823[M − H]^−^1.527,580,542.883446.08475; 461.10901; 461.2377350Iristectorin AC_23_H_24_O_12_5.228493.1335493.13381[M + H]^+^0.612,299,646.27395.1348; 367.067151BilobalideC_15_H_18_O_8_5.245325.0921325.09248[M − H]^−^1.2390,959,807.85193.12309; 219.10243; 325.0922252Quercetin 3- (6'''-p-coumarylglucosyl) (1- > 2) -rhamnosideC_36_H_36_O_18_5.271757.1976757.19752[M + H]^+^− 0.1371,061,325.91309.09674; 419.13394; 449.1079453p-Hydroxycinnamic acidC_9_H_8_O_3_5.271147.0432147.04392[M + H − H_2_O]^+^4.767,797,177.533106.83753; 118.39583; 119.04918546-HydroxyluteolinC_15_H_10_O_7_5.28303.0489303.04942[M + H]^+^1.6511,513,473.2303.04971, 304.0530755Iristectorigenin B 7-O-glucosideC_23_H_24_O_12_5.474537.1232537.12415[M + FA − H]^−^1.682,109,334.117313.0707; 135.044156FisetinC_15_H_10_O_6_5.506287.0538287.05438[M + H]^+^2.0914,880,378.23287.0548757Kaempferol 3- [ 6'''-p-coumarylglucosyl- (1- > 2) -rhamnoside]C_36_H_36_O_17_5.508741.2025741.20255[M + H]^+^061,965,455.64309.09683; 419.13367; 433.1130458DihydrokaempferolC_15_H_12_O_6_5.596287.0557287.05589[M-H]^−^0.73,532,072.15243.06668; 259.06097; 287.0560959(-)-LariciresinolC_20_H_24_O_6_5.749383.1457383.14614[M + Na]^+^1.043,174,197.907383.14664; 383.2021560MorinC_15_H_10_O_7_6.201301.0345301.03495[M − H]^−^1.6612,446,018.79178.99837; 301.03476; 301.1419161Ginkgolide BC_20_H_24_O_10_6.252423.1291423.1294[M − H]^−^0.71263,755,869.6367.13913; 395.1337; 423.1292762Ginkgolide AC_20_H_24_O_9_6.262453.1396453.13987[M + FA − H]^−^0.66692,269,705.2379.13992; 407.13483; 453.1405663Lirioresinol AC_22_H_26_O_8_6.298401.1585401.15926[M + H − H_2_O]^+^1.996,215,344.867371.1509; 383.1496; 401.159364ZedoarofuranC_15_H_20_O_4_6.769287.1246287.12503[M + Na]^+^1.391,730,123.657287.05545; 287.1252765KaempferolC_15_H_10_O_6_7.04285.0398285.04015[M − H]^−^1.410,014,194.53285.0402566TamarixetinC_16_H_12_O_7_7.223315.0506315.05079[M − H]^−^0.634,178,651.18177.0193; 301.071867Ginkgolide KC_20_H_22_O_9_7.789405.1181405.11862[M − H]^−^1.233,018,814.99377.12341; 405.11853; 406.1225968[6]-ShogaolC_17_H_2_4O_3_10.474275.1647275.16498[M − H]^−^1.092,158,949.157206.89798; 231.17526; 275.1650469PalmitamideC_16_H_33_NO12.695256.2629256.26318[M + H]^+^1.1715,138,303.45256.2632470StearamideC_18_H_37_NO13.951284.2936284.29424[M + H]^+^2.111,999,611.83284.29453
Molecular docking
Based on the relative quantification by UPLC-QE-MS, the 15 most abundant compounds in SXNI injection were selected for further investigation. These comprised five flavonoids and five terpenoid lactones (ginkgolides), which are recognized as the key characteristic constituents of SXNI, along with five other constituents (Figure S2). Molecular docking was performed to evaluate their binding potential to VEGF-A (PDB: 4QAF) and VEGF-C (PDB: 2X1W). The results indicated that all flavonoids and terpenoid lactones showed favorable binding affinities, with docking scores lower than -7.5 kcal/mol, whereas the remaining five compounds exhibited weak binding (Fig. 10A). Therefore, subsequent analysis focused on these ten compounds. Among the ginkgolides, ginkgolide B displayed the strongest binding to VEGF-A (docking score: − 8.0 kcal/mol), while ginkgolide A bound most effectively to VEGF-C (docking score: − 8.8 kcal/mol) (Fig. 10B). Within the flavonoids, quercetin 3-neohesperidoside showed the highest affinity for VEGF-A (− 10.2 kcal/mol), and rutin for VEGF-C (− 10.8 kcal/mol) (Fig. 10C). Consequently, ginkgolide A, ginkgolide B, quercetin 3-neohesperidoside, and rutin were preliminarily identified as the potential key active components in SXNI injection that may function by modulating VEGF-A and VEGF-C expression.Fig. 10. Molecular docking. A Binding Score of the Top 15 Components in the SXNI (red: terpenoid lactones; blue: flavonoids; black: other constituents). B Molecular docking visualizations (ginkgolide B, ginkgolide A). C Molecular docking visualizations (quercetin 3-neohesperidoside, rutin)
The key active components in SXNI promote VEGF-A and VEGF-C proteins release in Raw264.7 cells
In order to investigate the regulatory effects of ginkgolide A, ginkgolide B, rutin and quercetin 3-neohesperidoside on VEGF-A and VEGF-C expression in RAW264.7 cells, we first assessed their impact on RAW264.7 cell viability. The results showed that none of the four compounds exhibited significant toxicity at concentrations below 80 μM (Fig. 11A). We then examined their effects on Vegf-a and Vegf-c mRNA expression. As illustrated in Fig. 11B, C, each compound was found to significantly upregulate the transcriptional levels of Vegf-a and Vegf-c in Raw264.7 cells to varying degrees (^^P < 0.05, ^^P < 0.01, ^^P < 0.001). Further ELISA analysis revealed that, compared to the ctrl group, the levels of VEGF-A and VEGF-C protein secretion in the cell culture medium after treatment with the four compounds were elevated. Among them, ginkgolide A and ginkgolide B showed the most significant effects (^*^P < 0.05, ^^P < 0.01, Fig. 11D, E). These results suggest that ginkgolide A, ginkgolide B, rutin and quercetin 3-neohesperidoside are the active ingredients in SXNI that activate macrophages and stimulate the release of VEGF-A and VEGF-C.Fig. 11. The key active components in SXNI promote VEGF-A and VEGF-C proteins release in Raw264.7 cells. A Cell viability of Raw264.7 cells after ginkgolide A, ginkgolide B, rutin or quercetin 3-neohesperidoside treatment (n = 4). B, C Relative Vegf-a and Vegf-c mRNA levels in Raw264.7 cells after ginkgolide A, ginkgolide B, rutin or quercetin 3-neohesperidoside exposure (n = 3). D, E VEGF-A and VEGF-C proteins released by Raw264.7 cells after being treated with ginkgolide A, ginkgolide B, rutin or quercetin 3-neohesperidoside (n = 4). Data are expressed as mean ± SD. ^^P < 0.05, ^^P < 0.01, ^**^P < 0.001, versus the ctrl group; ns, no significant difference
Discussion
MI results in localized ischemia of the myocardium, leading to oxygen and nutrient deprivation, myocardial damage, and subsequent ventricular remodeling, cardiac insufficiency, and heart failure [37, 38]. Reestablishing blood flow to the affected area post-MI is crucial for tissue repair and preservation of cardiac function [39]. Despite significant advancements in MI treatment using pharmacological and interventional therapies, these approaches are often limited by contraindications, adverse effects, and high complication rates, which hinder their widespread clinical application [40]. TCM offers distinct advantages with its "multi-component, multi-target, multi-path" approach, having a key role in MI treatment and gaining global recognition [41]. SXNI, a Chinese herbal injection widely used in China for MI treatment, has demonstrated effective enhancement in long-term patient outcomes in clinical settings [42]. However, the pharmacological mechanisms underlying its therapeutic effects remain unclear. Herein, we investigated the cardioprotective effects of SXNI in MI rats and examined its potential mechanism involving angiogenesis and lymphangiogenesis.
The dose of SXNI for in vivo administration was determined using the following methodology. Based on the clinical dose of 20 mL per person per day for humans, and considering a conversion factor of 6.17 between human and rat body weights [43], doses of 1.05 mL/kg (low dose), 2.1 mL/kg (clinical equivalent dose), and 4.2 mL/kg (high dose) of SXNI were administered in this study. A rat MI model was induced by permanent ligation of the LAD to assess the cardioprotective potential of SXNI. After four weeks of treatment, except for the 1.05 mL/kg dose, SXNI enhanced cardiac function, reduced myocardial infarct size, attenuated cardiac fibrosis, and suppressed myocardial apoptosis in the MI model. These effects were demonstrated through echocardiography, TTC staining, Masson's trichrome staining, histopathology, and TUNEL staining. Thus, SXNI revealed the ability to enhance cardiac function post-MI and exert cardioprotective effects at both clinically equivalent doses (2.1 mL/kg) and high doses (4.2 mL/kg).
Therapeutic angiogenesis serves as a crucial strategy for revascularization in MI, essentially helping to restore blood flow to ischemic areas and salvaging injured myocardium [44]. CD31 staining, a marker specific to angiogenesis, was used in this in vivo study to assess microvascular density in the infarct border zone. SXNI significantly enhanced microvascular density when compared to the ischemia model. VEGF-A is a potent angiogenic factor and pivotal role in promoting angiogenesis and collateral vessel formation [45]. VEGFR-2 stands out as the primary receptor mediating the biological effects of VEGF-A. Activation of the AKT and ERK pathways via the VEGF-A/VEGFR-2 axis stimulates endothelial cell proliferation [34]. Our findings demonstrate that SXNI treatment significantly increased VEGF-A expression levels in cardiac tissue and plasma following MI. Moreover, western blot analysis revealed that SXNI activated the VEGF-A/VEGFR-2 signaling pathway, enhancing the expression of p-AKT and p-ERK, thus facilitating a pro-angiogenic effect.
In MI treatment, research has traditionally focused on the vascular system while overlooking the regulatory role of the lymphatic system, which is increasingly recognized as crucial [46]. Recent studies highlight that impaired lymphatic vessels post-MI can lead to adverse cardiac remodeling, including lymphedema, heightened inflammation, and fibrosis [47]. Pharmacological studies have underscored the importance of lymphangiogenesis in cardiac function recovery following cardiac injury [33]. SXNI, known for its potential to promote lymphangiogenesis and enhance silica clearance in the lung, has garnered attention [21]. However, its specific impact on cardiac lymphangiogenesis post-MI remains unclear. LYVE-1 immunohistochemical staining, a specific marker of lymphangiogenesis, was used in this study to assess lymphatic vessel density in the infarct border zone. Our findings indicate that SXNI treatment significantly enhances lymphatic vessel density compared to the ischemic model.
VEGF-C, a principal growth factor in lymphangiogenesis, operates through the VEGF-C/VEGFR3 signaling axis. Previous studies using microparticle-coated VEGF-C protein injections into the heart have demonstrated substantial promotion of lymphatic vessel formation and mitigation of cardiac damage [28]. In our study, SXNI treatment notably increased VEGF-C expression levels in myocardial tissue and plasma post-MI. This enhancement activated the VEGF-C/VEGFR3 pathway, thereby promoting lymphangiogenesis. Furthermore, our investigation revealed that SXNI treatment was associated with significantly reduced cardiac water content compared to the model group. Similarly, levels of inflammatory cytokines IL-1β, TNF-α, and IL-6 in the serum of SXNI-treated rats were markedly lower than those in the model group. These findings indicate that SXNI may mitigate cardiac edema and suppress inflammatory factor release in cardiac tissue by fostering lymphangiogenesis following MI.
The restoration of collateral circulation is pivotal in restoring adequate oxygen and nutrient supply to ischemic areas, thereby promoting myocardial viability [48]. While therapeutic angiogenesis typically enhances VEGF-A expression to stimulate angiogenesis, this increase is often associated with heightened vascular permeability and tissue edema [49]. Therefore, mitigating myocardial tissue edema is a key consideration in VEGF-A-based therapies. A recent study used HepNP to co-deliver VEGF-C and VEGF-A proteins to the heart, effectively promoting collateral circulation, reducing edema, and enhancing cardiac function [8]. Our study revealed that SXNI concurrently enhances the expression of both VEGF-A and VEGF-C proteins following MI. This dual effect activates the VEGF-A/VEGFR2 signaling pathway for angiogenesis and the VEGF-C/VEGFR3 pathway for lymphangiogenesis. Consequently, SXNI facilitates enhanced blood flow supply while mitigating cardiac edema and inflammation, thereby exerting a multifaceted cardioprotective effect post-MI.
As integral components in the innate immune response, macrophages play a crucial role in cardiac repair following MI. They are primary producers of VEGF-A and VEGF-C, which promote angiogenesis and lymphangiogenesis through cytokine secretion [50]. To delve deeper into the indirect pharmacological mechanism by which SXNI regulates these processes, we conducted a series of in vitro assays. Our findings demonstrate that SXNI treatment increases the release of VEGF-A and VEGF-C from Raw264.7 cells. Subsequently, we evaluated the effects of SXNI on HUVECs and SVEC4-10 cells, specifically examining whether these effects are indirectly mediated by macrophage activation. Notably, SXNI alone did not directly enhance the biological activities of HUVECs or SVEC4-10 cells. However, supernatants derived from SXNI-treated Raw264.7 cells significantly enhanced the proliferation, migration, and tube formation capabilities of HUVECs and SVEC4-10 cells, indicating that SXNI acts indirectly through macrophage-derived factors. Further mechanistic studies revealed that these macrophage-conditioned supernatants activated the p-VEGFR-2, p-AKT, and p-ERK signaling pathways in HUVECs, as well as VEGFR3, p-AKT, and p-ERK in SVEC4-10 cells, further delineating the indirect signaling cascade initiated by SXNI via macrophage modulation.
Traditional Chinese medicine serves as an important source of natural products, which contain structurally novel compounds with potent biological activities. In this study, we used liquid chromatography-mass spectrometry to identify 70 chemical constituents in SXNI, then performed molecular docking analyses to evaluate their binding affinities towards VEGF-A and VEGF-C proteins. Flavonol glycosides and terpene lactones were identified as the major and characteristic constituents of SXNI among these. Of these, ginkgolide A, ginkgolide B, rutin and quercetin 3-neohesperidoside were found to have strong binding interactions with both VEGF-A and VEGF-C. Furthermore, subsequent cell-based experiments demonstrated that these compounds increased the mRNA and protein expression levels of VEGF-A and VEGF-C in Raw264.7 cells. Therefore, our findings highlight the role of SXNI in promoting angiogenic and lymphangiogenic activities via macrophage-mediated pathways. Ginkgolide A, ginkgolide B, rutin and quercetin 3-neohesperidoside were identified as the key bioactive components in SXNI responsible for activating macrophages and subsequently releasing VEGF-A and VEGF-C (Fig. 12). Given the predictive nature of molecular docking, further experimental validation, such as by surface plasmon resonance or competitive binding assays—is necessary in future studies. Meanwhile, macrophage-depleted animal models should be employed to confirm the essential role of macrophages in vivo. Together, these integrated strategies will further elucidate the precise mechanism of Shuxuening Injection and strengthen the translational potential of our findings.Fig. 12. Schematic illustration of SXNI alleviating cardiac remodeling after MI by promoting angiogenesis and lymphangiogenesis
Conclusion
This study demonstrates that SXNI exerts its cardioprotective effects post-MI through an indirect pharmacological mechanism. Rather than acting directly on endothelial cells, SXNI activates macrophages to secrete VEGFs, such as VEGF-A and VEGF-C, which subsequently activate VEGFR2- and VEGFR3-mediated ERK/AKT pathways in endothelial cells, promoting angiogenesis and lymphangiogenesis to improve cardiac function and attenuate fibrosis, edema, and inflammation. Furthermore, we identify ginkgolide A, ginkgolide B, rutin, and quercetin 3-neohesperidoside as the key SXNI components that activate macrophages. Collectively, SXNI represents a promising therapeutic that acts indirectly via immune modulation to promote vascular and lymphatic repair in MI.
Supplementary Information
Additional file1 (DOCX 865 kb)Additional file2 (DOCX 1040 kb)
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