Male C57BL/6J mice were provided by Beijing Huafukang Bioscience Co., Ltd. (Beijing, China; license number: SCXK [Jing] 2019-0008). Forty mice after 1 week of acclimatization, under anesthesia with 1% isoflurane, a 3-mm incision was made at the proximal sternum to fully expose the aortic arch. A blunt 27-gauge needle was positioned between the two carotid arteries, directly over the aortic arch. Using 7-0 silk suture, the aortic arch was ligated against the needle at a site immediately distal to the brachiocephalic artery. The needle was then promptly removed, creating a reproducible TAC. Following the procedure, the sternum and skin were closed with 6-0 polypropylene sutures, and the mice were placed on a pre-warmed heating pad until full recovery from anesthesia. Sham-operated animals underwent the identical surgical exposure without aortic ligation. Beginning on postoperative day 2, the drug concentration is based on previous literature [11, 12], mice assigned to the high-dose group (APS-H) and low-dose group (APS-L) received intragastric administration of APS at 50 mg/kg/day and 100 mg/kg/day, respectively (APS: Solarbio, China, cat. no.AGV 7970, purity $\ge$90%); the remaining groups received an equivalent volume of purified water. Treatment continued for 4 consecutive weeks. At 4 weeks post-surgery, mice were re-anesthetized using a precision gas anesthesia system, with isoflurane concentration titrated to 2.5% $\pm$ 0.5% for induction and maintained at 1.8% $\pm$ 0.3% for imaging, while oxygen flow was maintained at 1 L/min. Following transthoracic echocardiographic assessment, animals were euthanized via cervical dislocation. Freshly excised hearts were immediately weighed and processed for downstream analyses, including histopathology and western blotting.

At 4 weeks post-surgery, cardiac function was assessed using a high-resolution small-animal ultrasound system (The manufacturers and production addresses of the equipment used are listed in Table 1). Measured parameters included left ventricular ejection fraction (LVEF), left ventricular fractional shortening (LVFS), left ventricular end-diastolic internal diameter (LVIDd), left ventricular end-systolic internal diameter (LVIDs), left atrial diameter (LAD), and left atrial diastolic area (LA diastolic area).

Cardiac tissues were paraffin-embedded, sectioned, and subjected to Masson’s trichrome staining according to the instructions provided by the manufacturer’s instructions (The manufacturers, production addresses, and batch numbers of the kits used are shown in Table 1). Images were acquired using an upright microscope under identical settings (magnification: $\times$20). For each animal, at least three sections were analyzed, and 5–10 random fields per section were selected. ImageJ software (Wayne Rasband and contributors National Institutes of Health, USA, Java 1.8.0 345, http://imagej.org) was used to separate the color channels, and a uniform threshold for the collagen-positive blue channel was set based on control and positive reference samples. Batch processing was applied to all images.

Paraffin-embedded sections were subjected to deparaffinization, hydration, and antigen retrieval before immunohistochemical staining, which was carried out using a commercially available kit (The manufacturers, production addresses, and batch numbers of the kits used are shown in Table 1). Expression of collagen I (Col-I) was detected under a light microscope, and quantitative evaluation was subsequently performed.

Cardiac sections were first fixed and permeabilized, then blocked with 5% bovine serum albumin. After overnight exposure to anti-Col-I antibodies, samples were incubated with fluorophore-conjugated secondary antibodies and counterstained with DAPI. Fluorescence images were acquired under identical conditions at $\times$40 magnification. The average signal intensity was measured using ImageJ, normalized to myocardial area, and the percentage of positive area was compared with the control group (The manufacturers, batch numbers, and preparation ratios of the antibodies used are shown in Table 1).

H9C2 rat cardiomyoblasts (ATCC® CRL-1446™) were obtained from ATCC (Manassas, VA, USA). Cell lines were authenticated by short tandem repeat profiling, regularly screened for mycoplasma contamination with the MycoAlert™ kit (Lonza, Switzerland), and cross-referenced with ICLAC and Cellosaurus databases. Cells were maintained in high-glucose DMEM supplemented with 10% fetal bovine serum and 1% penicillin–streptomycin. After reaching ~50–60% confluence, cultures were serum-deprived for 24 h. Hypertrophy was induced by Ang II (1 $\times$ 10^-5 mmol/L) for 24 h. To examine the involvement of TGF-$\beta$₁/Smad3 signaling, cells were exposed to the agonist SRI-011381 (10 µM), either alone or in combination with APS (600 µg/mL).

Cells were seeded into 96-well plates and incubated for 24 hours (1 $\times$ 10⁴ cells per well), followed by treatment with varying concentrations of Ang II for an additional 24 hours. Subsequently, 10 µL of CCK-8 solution was added to each well and incubated in the incubator for 2 hours. Absorbance was then measured at 450 nm using a dual-wavelength method, and the data were analyzed with GraphPad Prism 8.4.0 (GraphPad Software Inc., San Diego, CA, USA).

Proteins were extracted from ventricular tissues or cultured cells, quantified, and separated by SDS-PAGE. After transfer onto PVDF membranes, blocking was performed with 5% non-fat milk. Membranes were incubated with primary antibodies overnight, followed by HRP-conjugated secondary antibodies (The manufacturers, batch numbers, and preparation ratios of the antibodies used are shown in Table 1). Protein bands were visualized using enhanced chemiluminescence and analyzed with ImageJ software.

Data analysis was conducted with GraphPad Prism 8.4.0 (GraphPad Software Inc., San Diego, CA, USA). One-way ANOVA followed by Tukey’s multiple comparison test was applied to determine intergroup differences. A two-tailed p-value 0.05 was considered statistically significant.

Four weeks after TAC surgery, compared with controls, mice displayed significant cardiac impairment. TAC mice showed marked declines in LVEF and LVFS, accompanied by enlarged LVIDd, LVIDs, LAD, and LA diastolic area, consistent with ventricular remodeling, and the heart weight/body weight (HW/BW) ratio was significantly elevated. These changes are consistent with the typical features of HCM. For example, studies have shown that the TAC model leads to a significant reduction in EF, with FS decreasing from 53.2% to 32.3%, indicating impaired cardiac contractile function [13]. In addition, TAC-induced pressure overload can trigger cardiac remodeling, manifested as ventricular wall thickening and chamber dilation. These alterations correspond well with the imaging characteristics observed in clinical HCM [14]. Administration of APS ameliorated these abnormalities in a dose-dependent manner. The APS-H exhibited notable improvements in systolic performance and reduced ventricular dilation, whereas the APS-L showed moderate but significant benefits. These findings highlight the cardioprotective action of APS against pressure overload induced dysfunction (Fig. 1A–I).

Fig. 1.

Astragalus polysaccharide (APS) improves cardiac function in transverse aortic constriction (TAC) mice. (A) Heart photo (scale bar = 1 mm). (B) Heart weight/body weight (HW/BW). (C) Transthoracic echocardiography. (D) Left ventricular ejection fraction (LVEF). (E) End-systolic left ventricular diameter (LVIDs). (F) Eend-diastolic left ventricular diameter (LVIDd). (G) Mean left atrial straight diameter (LAD). (H) Left ventricular short axis shortening (LVFS). (I) Left atrial diastolic area (LA diastolic area). ns, no statistical difference. APS-L, low-dose APS, 50 mg/kg/day; APS-H, high-dose APS, 100 mg/kg/day. n = 6.

Masson’s staining demonstrated a robust increase in collagen accumulation in the TAC group, confirming extensive fibrotic remodeling. Quantitative analysis revealed a substantial rise in collagen volume fraction relative to controls. Treatment with APS effectively reduced collagen deposition, with the most profound suppression observed in APS-H mice. Consistently, Col-I expression, as assessed by immunohistochemistry and immunofluorescence, was significantly diminished following APS treatment, indicating its potent antifibrotic effect (Fig. 2A–F).

Fig. 2.

APS attenuates TAC-induced myocardial fibrosis. (A) Ventricular Masson staining. (B) Collagen I (Col-I) is a representative immunohistochemical staining of the ventricle. (C) Statistical analysis of the atrial fibrosis area (scale bar = 50 µm). (D) Col-I average histochemical analysis. (E) Col-I (red) and DAPI (blue) representative ventricular immunofluorescence staining (scale bar = 100 µm). (F) Col-I average fluorescence intensity analysis. n = 6.

Western blot analysis revealed that TAC-induced remodeling was associated with increased myocardial expression of TGF-$\beta$₁, phosphorylated Smad3 (p-Smad3), and Col-I, while total Smad3 remained unchanged. APS administration markedly suppressed TGF-$\beta$₁ and p-Smad3 levels, along with Col-I expression. The reduction was most pronounced in APS-H animals, suggesting that APS exerts its antifibrotic activity largely through inhibition of TGF-$\beta$₁/Smad3 signaling (Fig. 3A–E).

Fig. 3.

APS modulates the expression of growth factor-$\beta$₁ (TGF-$\beta$₁)/Smad3 pathway-related proteins. (A) Western blotting detecting the expression of TGF-$\beta$₁, Smad3, phosphorylated Smad3 (p-Smad3), and Col-I in ventricular tissue. (B–E) Western blotting quantitative analysis. ns, no statistical difference. n = 6.

With increasing concentrations of Ang II, cell viability decreased significantly. At 10^-5 mmol/L, cell viability was approximately 70% of the control, which is commonly used as the experimental concentration; at 10^-4 mmol/L, notable cell damage occurred, indicating potential toxicity. Therefore, 10^-5 mmol/L was selected for subsequent experiments (Fig. 4A). Exposure of H9C2 cardiomyocytes to Ang II markedly upregulated the expression of TGF-$\beta$₁, p-Smad3, and Col-I, whereas co-treatment with APS attenuated these changes. APS alone had no detectable effect under basal conditions (Fig. 4B–F). Ang II treatment also significantly increased cell size, confirming successful induction of a hypertrophic phenotype, while APS treatment partially reduced cell size (Fig. 4G,H). Immunofluorescence further confirmed the downregulation of Col-I in the APS-treated group, reinforcing its role in counteracting Ang II–induced fibrosis-related remodeling at the cellular level (Fig. 4I,J).

Fig. 4.

APS suppresses angiotensin IIstimulated (Ang II)-induced hypertrophy in H9C2 cells. (A) Cell counting kit-8 (CCK8) assay of cell viability at different concentrations of Ang II. (B) Western blotting detecting the expression of TGF-$\beta$1, Smad3, p-Smad3, and Col-I in H9C2 cells. (C–F) Western blotting quantitative analysis. (G) Wheat Germ Agglutinin (WGA) staining, WGA (red) and DAPI (blue) representative ventricular immunofluorescence staining (scale bar = 100 µm). (H) Cell size analysis. (I) Col-I (red) and DAPI (blue) representative ventricular immunofluorescence staining (scale bar = 100 µm). (J) Col-I average fluorescence intensity analysis. ns, no statistical difference. n = 6.

To further confirm pathway specificity, Ang II-stimulated cells were co-incubated with the TGF-$\beta$₁/Smad3 agonist SRI-011381. This intervention enhanced TGF-$\beta$₁, p-Smad3, and Col-I expression and abolished the inhibitory effects of APS. Similarly, the reduction of Col-I fluorescence intensity by APS was no longer evident in the presence of the agonist. These results strongly support the conclusion that APS-mediated suppression of myocardial fibrosis relies on regulation of the TGF-$\beta$₁/Smad3 signaling cascade (Fig. 5A–G).

Fig. 5.

APS exerts antifibrotic-Fibrotic effects via the TGF-$\beta$₁/Smad3 pathway. (A) Western blotting detecting the expression of TGF-$\beta$₁, Smad3, p-Smad3, and Col-I in H9C2 cells. (B–E) Western blotting quantitative analysis. (F) Col-I (red) and DAPI (blue) representative ventricular immunofluorescence staining (scale bar = 100 µm). (G) Col-I average fluorescence intensity analysis. ns, no statistical difference. n = 6.