Senkyunolide I and Danshensu in combination ameliorate post-MI myocardial fibrosis by regulating TGF-β1/PAI-1 in mice and heart organoids

FIGURE 1. The effect of GXNI on echocardiographic characteristics of cardiac function and coronary blood flow in MI mice. Cardiac function and coronary blood flow were assessed in each group of mice on days 1, 14, and 28 after permanent ligation of the LAD artery. (A) Representative echocardiographic images of the sham, model, GXNI 1 mL/kg, GXNI 3 mL/kg, GXNI 9 mL/kg, and MP (1 mL/kg)-treated groups. (B) Quantitation of EF%. (C) Quantitation of FS %. (D) Representative echocardiography images of the coronary blood flow determined in color Doppler mode of each group. (E) Quantification of AV peak velocity. (F) Quantification of AV peak pressure. (G) Quantification of AoV VTI. Values were expressed as mean ± SD (n = 5). ### P < 0.001 vs. sham, *P < 0.05, **P < 0.01, ***P < 0.001 vs. model (EF, ejection fraction; FS, fractional shortening; AV, aortic valve; AoV VTI, aorta velocity–time integral mean velocity).

FIGURE 2. GXNI reduced heart weight, infarct area, and collagen deposition of MI mice. The heart tissues were removed after 28 days for pathological and statistical analyses, including sham, MI model, MP, GXNI 1 mL/kg, GXNI 3 mL/kg, and GXNI 9 mL/kg-treated mice. (A) Representative images of heart samples from each group. (B) Representative images of H&E staining and Masson staining results, where myocardial damage is indicated by yellow arrows and collagen deposition is indicated by blue arrows. The upper panel is panoramic scan images of myocardial tissue in cross-section. Normal myocardial tissue was red, while the infarcted area was white. (C) Quantification of heart weight in each group. (D) Quantification of infarct volume in each group. (E) Quantification of the area of collagen deposition in each group (n = 5). (F) Representative images of α-SMA immunofluorescence staining in non-infarcted areas and infarcted areas. (G) Quantification of α-SMA positivity in mouse heart tissue. (H) Gene expression of α-SMA in mouse heart tissue. (I) Gene expression of Col1a1 in mouse heart tissue. (J) Gene expression of Col3a1 in mouse heart tissue. Data were expressed as mean ± SD. ### P < 0.001…

FIGURE 3. TGF-β1/PAI-1 is a key target of GXNI to inhibit myocardial fibrosis after MI. (A) Venn diagram of the “myocardial infarction” and “myocardial fibrosis” target libraries. (B) Top 10 typical pathways of disease through IPA core analysis. (C) Fibrosis-related pathways. (D) Venn diagram of “myocardial infarction” target library, “myocardial fibrosis” target library, and “cardiac GXNI vs. MI” transcriptome dataset. (E) Protein-protein interaction network diagram. (F) Representative WB image of PAI-1 and GAPGH for myocardial tissue in sham, model, and 3 mL/kg GXNI groups. (G) Quantification of PAI-1 WB results (n = 3). (H) The Tgf-β1 mRNA expression levels (n = 3). (I) Representative images of IHC staining of PAI-1 and TGF-β1 for mouse myocardial tissue in sham, model, and 3 mL/kg GXNI groups. (J–K) Quantification of PAI-1 and TGF-β1 IHC results (n = 3). (L) Immunofluorescence staining of TGF-β1 in the mouse heart. (M) Immunofluorescence staining of PAI-1 in the mouse heart. (N) Quantification of TGF-β1 positive area in the mouse heart (n = 3). (O) Quantification of TGF-β1 positive area in the mouse heart (n = 3). Data were expressed as mean ± SD. Data were expressed as mean ± SD. ## P…

FIGURE 4. Ingredients in GXNI that bind to PAI-1. Structural-based binding prediction of GXNI ingredients to PAI-1 by molecular docking. (A) Chemical structures of the ten main active ingredients of GXNI. (B) Key GXNI active ingredient-disease target network. Nodes are color-coded to indicate their origin: Danshen ingredients are blue, Chuanxiong ingredients are yellow, and disease targets are pink. The size of each node is proportional to its degree value, reflecting the number of target interactions; a larger oval signifies that an active ingredient acts on a greater number of disease targets. TGF-B1 and SERPINE1 (PAI-1) are highlighted in red. (C) Relative affinity ranking of the ten main active ingredients of GXNI with PAI-1. (D) Molecular docking site map of the ten main active ingredients of GXNI and PAI-1. Tiplaxtinin, a PAI-1 inhibitor, served as a control. (E) Representative WB blots of cellular thermal shift assay. (F) The binding intensity curve of CETSA.

FIGURE 5. Effects of GXNI and DSS + SENI on TGF-β1-induced activation, migration, and proliferation of CFs. (A) Representative images of Sirius Red staining. (B) Quantification of collagen content (n = 3). (C) Changes in the migration of primary cardiac fibroblasts. (D) Representative images of fluorescence intensity of α-SMA. (E) 24 h fibroblast mobility (n = 3). (F) Quantification of α-SMA immunofluorescence staining. (G) Quantification of the number of primary cardiac fibroblast nuclei (n = 3). (H) Representative stained images of the nuclei (blue by Hoechst), the TGF-β1 protein (green), and the PAI-1 protein (yellow). (I) Quantification of the average fluorescence intensity of TGF-β1 (n = 3). (J) Quantification of the average fluorescence intensity of PAI-1 (n = 3). Data were expressed as mean ± SD. ### P < 0.001 vs. control, ** P < 0.01, *** P < 0.001 vs. TGF-β1. Scale bar = 100 µm.

FIGURE 6. DSS SENI protects against myocardial fibrosis by regulating the TGF-β1/PAI-1 pathway in cryoinjury organoid. (A) Representative images of PI flow cytometry. (B) Representative images of fluorescent staining of cardiac organoids for Calcein AM and PI in each group. (C) PI-positive cells for heart organoids by flow cytometry analysis (D) Quantification of the cell viability, (E) average fluorescence intensity of calcein AM, and (F) average fluorescence intensity of PI (n = 6). (G,H) Quantification of the fluorescence intensity of α-SMA and PAI-1 (n = 6). (I) Representative images of immunofluorescence staining of α-SMA and PAI-1 in cardiac organoids. Representative chart of Sirius Red staining. (J) Representative images of Masson and Sirius Red staining of organoids. (K,L) Quantification of the collagen deposition area and Sirius positive area (n = 6). (M) Quantification of the average fluorescence intensity of PAI-1 in each group (n = 6). (N) Representative images of immunofluorescence staining of PAI-1 in organoids. (O) Representative gene expression by qPCR. (P) Representative images of immunofluorescence staining of TGF-β1 in organoids. (Q) Quantification of the average…

FIGURE 7. Schematic diagram of the mechanism by which GXNI modulates TGF-β1/PAI-1 to improve myocardial fibrosis after myocardial infarction in mice.