In this study, 8-week-old C57BL/6 mice purchased from Jackson Laboratory (Sacramento, CA) were treated as wild-type (WT). We treated the mice with Ang II (1,000 ng/kg/min) or saline for 14 days. The hypertensive myocardial remodeling models and controls were established using osmotic micropumps (Alzert Model 1002, DURECT, Cupertino, CA). Ginaton injection was purchased from Chi Sheng Chemical Cooperation (Taiwan, China). To investigate the potential protective effects of Ginaton, we administered Ginaton (300 mg/kg/day) to the mice intragastrically the day before surgery, at a dose according to that reported previously (Sun et al., 2015). Phosphate-buffered saline (PBS) treatment was used as a control. All animal experiments were carried out in accordance with the National Institutes of Health (NIH) Guidelines for the Care and Use of Laboratory Animals, which were approved by the Dalian Medical University Animal Committee.

We used a tail-cuff device (BP-98, Softron, Japan) to record systolic blood pressure (SBP) and heart rate in all mice. We placed the mice on a retainer, completely exposing the tail, and then placed the tail-cuff device to measure their SBP and heart rate. Cardiac function was measured in each mouse using a 30 MHz probe (Vevo 1100 system, VisualSonics, Toronto, Canada). By measuring the echocardiography of each mouse, we recorded the end-diastolic and end-systolic left ventricular anterior wall (LVAW) thickness, left ventricular posterior wall (LVPW) thickness, ventricular inner diameter (ID), LV ejection fraction (EF%), and LV fractional shortening (FS%).

Heart tissue was fixed in 4% POM for >24 h. Part of the heart tissue was paraffin-embedded, while the other part was OCT-embedded. Masson’s trichrome and hematoxylin and eosin (H&E) staining were performed on paraffin sections (4 μm). The OCT sections (8 μm) were treated with wheat germ agglutinin (WGA). Cardiac sections were stained with anti-α-SMA (ab124964, Abcam) by immunohistochemistry at 4°C overnight. The next day, after washing with PBS, the sections were incubated with the secondary antibody and DAB substrate. The color reaction was stopped with ddH2O, and the sections were incubated with hematoxylin. Frozen sections or cells were fixed with 4% POM for 15 min at room temperature and then incubated with anti-CD68 (ab283654, Abcam), anti-CD206 (ab300621, Abcam), or anti-iNOS (ab283655, Abcam) at 4°C overnight. The next day, after washing with PBS, the sections were incubated with fluorescently labeled antibodies at room temperature for 30 min and then with DAPI at room temperature for 3 min. Pictures of the sections were taken at ×100/×200 magnification on a fluorescence microscope (Olympus, BX53, Japan).

Total RNA was extracted from fresh heart tissue or cells. cDNA (1 μg) was obtained using PrimeScript RT reagent (Takara, Japan). Quantitative real-time PCR was performed with an SYBR Green Premix Pro Taq HS qPCR Kit (AG11701). All mRNA levels were normalized to GAPDH by ΔΔCt analysis.

Fresh heart tissue was added to RIPA lysis buffer to obtain total protein. Protein (25 μg) was subjected to SDS-PAGE and transferred to polyvinylidene fluoride membranes. The membranes were blocked with skim milk for 30 min at room temperature and then incubated with primary antibodies at 4°C overnight. The next day, after washing with TBST, the membranes were incubated with conjugated secondary antibodies (1:3000, CST) at room temperature for 1 h, and the bands were visualized by chemiluminescence. The main antibodies used in this study were: anti-TGF-β (ab215715, Abcam), anti-p-Smad2 (ab280888, Abcam), anti-Smad2 (ab33875, Abcam), anti-p-AKT (ab38449, Abcam), anti-AKT (orb11276, Biorbyt), anti-p-ERK1/2 (ab214036, Abcam), anti-ERK1/2 (ab184699, Abcam), anti-p-IKKα (ab138426, Abcam), anti-IKKα (ab32041, Abcam), anti-p-P65 (ab76302, Abcam), anti-P65 (ab16502, Abcam), and anti-GAPDH (ab8245, Abcam).

Macrophages were obtained from the tibias and femurs of WT mice and cultured in 1640 medium (Meilunbio, MA0215). Human umbilical vein endothelial cells (HUVECs) were cultured in ECM. Proclones from macrophages to adherent HUVECs were distributed as previously described (Yin et al., 2022). We pretreated HUVECs with Ginaton (100 μg/ml) or PBS for 4 h and then with Ang II (100 nM) or saline for 24 h. Macrophages were labeled with PKH-26 fluorescent dye and added to HUVECs at a ratio of 10:1. After incubation for 1 h, the non-adherent cells were washed with PBS, while the adherent cells were photographed by microscopy.

HUVECs were cultured and treated as described in the adhesion experiments. Macrophages (5 × 10⁴) were added to the upper chamber of a 24-well Transwell plate (8 μm pore, Coning), and HUVEC-conditioned media were added to the lower chamber of the plate. After 24 h of incubation, the migrated cells were fixed, stained with DAPI, and observed by microscopy.

Data are presented as means ± SD. Statistical analysis was performed using GraphPad Prism software. For statistical comparison, a one-way ANOVA was used, followed by Dunnett’s multiple comparison tests with the control group. Student’s unpaired t-tests were used to compare the two groups. p <0.05 was considered statistically significant.

As shown in Figure 1A, WT mice were treated with Ginaton (300 mg/kg) and Ang II (1,000 ng/kg/min) for 14 days. PBS was used as the control. After 14 days of Ang II infusion, the SBP in the Ginaton-treated group was significantly lower than that in the PBS-treated group. However, after Ang II infusion, heart rates were similar in the Ginaton and PBS treatment groups (Figure 1B). Echocardiography showed enhanced cardiac function 14 days after Ang II infusion, with increased EF% and FS% in the PBS-treated group, while treatment with Ginaton effectively mitigated this response (Figures 1C, D, Supplementary Table S1).

Next, we determined the role of Ginaton in Ang II-induced cardiac hypertrophy. After 14 days of Ang II infusion, PBS-treated mice exhibited the characteristics of myocardial hypertrophy, including increased left ventricular thickness, heart weight/body weight (HW/BW), heart weight/tibial length (HW/TL), and increased cardiomyocyte cross-sectional area; however, these effects were mitigated by Ginaton (Figures 2A, B). Ginaton also reduced the expression of hypertrophy markers (ANF and BNP) after Ang II infusion (Figure 2C). The primer sequences are listed in Supplementary Table S2. As shown in Figure 2D, Ginaton downregulated the Ang II-induced activation of AKT and ERK1/2 expression. Therefore, Ginaton attenuated the Ang II-induced cardiac hypertrophy response.

Masson’s trichrome staining to investigate the role of Ginaton in Ang II-induced cardiac fibrosis showed that Ginaton treatment significantly reduced collagen deposition in the Ang II-infused heart compared to the PBS group (Figure 3A). Similarly, the number of α-SMA-positive myofibroblasts and the expression levels of α-SMA, collagen I, and collagen III mRNA were downregulated in Ginaton-treated mice compared to the PBS group (Figures 3B, C). As shown in Figure 3D, Ang II infusion induced increased TGF-β and Smad2 protein expression, while Ginaton treatment alleviated this response. These results suggested that Ginaton participates in Ang II-induced myocardial fibrosis through the TGF-β-Smad2 pathway.

To investigate the expression of different macrophage phenotypes in Ang II-infused hearts and the mechanism by which Ginaton improved Ang II-induced cardiac remodeling, we co-stained with the CD68 macrophage biomarker and the iNOS M1 phenotype macrophage biomarker. Immunofluorescent staining revealed that Ang II infusion induced high expression of iNOS⁺ M1 phenotype macrophages and that Ginaton reduced this Ang II-induced increase (Figure 4A). Furthermore, co-staining for the CD68 macrophage biomarker and the CD206 M2 phenotype macrophage biomarker showed no significant difference between Ang II infusion and Ginaton treatment on CD206⁺ M2 phenotype macrophages (Figure 4B). Similarly, Ang II infusion increased the mRNA levels of molecules associated with M1 phenotypic expression, including IL-1β, IL-6, TNF-α, and MCP-1, while Ginaton rescued this Ang II-induced inflammatory reaction. The levels of M2 phenotypic expression molecules, including Arg1, Ym1, and IL-10 mRNA levels, did not differ between the Ang II infusion and Ginaton treatment groups (Figures 4C, D). Moreover, IKKα and p65 phosphorylation were reduced in the Ginaton-treated group compared to the PBS-treated group (Figure 4E). These results suggested that Ginaton relieved Ang II-induced cardiac inflammation via the accumulation of M1 phenotype macrophages in the heart.

To confirm the effect of Ginaton on the activation of M1 phenotype macrophages induced by Ang II, we pretreated macrophages with Ginaton (100 μg/ml) or PBS for 4 h and with Ang II (100 nM) or saline for 24 h. We then co-stained the cells with the macrophage biomarker CD68 and the M1 phenotype macrophage biomarker iNOS. Immunofluorescence staining showed that Ang II treatment induced high expression of iNOS⁺ M1 phenotype macrophages and that Ginaton reduced the increase in iNOS⁺ M1 phenotype macrophages induced by Ang II in vitro (Figure 5A). Moreover, Ginaton reduced Ang II-induced mRNA expression of the M1 phenotype molecules IL-1β, IL-6, TNF-α, and MCP-1 (Figure 5B). Neither immunofluorescence staining nor qPCR results showed differences in M2 phenotype macrophages between the Ginaton and Ang II treatment groups (Figures 5C, D).

Coculture experiments of macrophages with HUVECs and Transwell assays showed that Ang II treatment increased macrophage adhesion and migration to HUVECs, an effect that was inhibited by Ginaton (Figures 6A, B). The results showed that Ginaton could reduce the adhesion and migration of macrophages to HUVECs.