Zhilong Huoxue Tongyu capsule alleviates myocardial fibrosis by improving endothelial cell dysfunction

Figures

• 

  Fig. 1. Dysfunction of EC was induced during myocardial fibrosis (A, B) UMAP showing the expression of CD31 and α-SMA/FSP1 in different pathologies. (C) Quantitative changes of CD31+HLA2+ cell subsets between diseased and normal groups, as determined by single-cell sequencing. (D) Quantification of the frequencies of CD4+T cell in two different groups. All of the results are shown as the mean ± S.D.; ∗P < 0.05; ∗∗P < 0.01.

• 

  Fig. 2. Qualitative UPLC-HR-MS analysis of ZL capsule (A) UPLC-HR-MS chromatogram of ZL capsule in negative ion mode. (B–C) UPLC-HR-MS chromatogram of 12 standards, namely, salidroside (1), chlorogenic acid (2), L-epicatechin (3), calycosin-7-glucoside (4), ononin (5), calycosin (6), formononetin (7), astragaloside A (8), astragaloside III (9), wogonin (10), astragaloside II (11), and astragaloside I (12). (D) Discovery of the eight targets of ZL capsule that are closely related to EC dysfunction.

• 

  Fig. 3. The establishment of ISO-induced MF mouse model was successful. (A) Process of establishing the present model of mouse myocardium. (B) Histological analysis of myocardial fibrosis. H&E, Sirius Red, and Masson staining results of cardiac tissue (n = 4). Scale bars: 200 μm. (C) Immunohistochemical and immunofluorescence analyses of markers (α-SMA, collagen-I, and collagen-III) related to fibrosis (n = 4). Scale bars: 100 μm. (D) Quantification of relative proportion of α-SMA, collagen-I, and collagen-III cells per visual field for sham and ISO-model (n = 4). All of the results are shown as the mean ± S.D.; ∗P < 0.05; ∗∗P < 0.01.

• 

  Fig. 4. Degree of EndMT and the extent of endothelial MHC-II antigen presentation to T cell are enhanced during myocardial fibrosis. (A) Co-staining of ISO-model or control myocardial sections for the EC marker CD31 and the mesenchymal marker α-SMA (n = 4). Scale bars: 100 μm. (B) Co-staining of ISO-model or control myocardial sections for CD31 and the mesenchymal marker FSP1 (n = 4). Scale bars: 100 μm. (C) Quantification of the numbers of CD31+α-SMA+ and CD31+FSP1+ double-positive cells per visual field. (D) Co-staining of ISO-model or control myocardial sections for CD31 and the MHC class II marker MHC-II (n = 4). Scale bars: 100 μm. (E) Immunofluorescent staining against CD4 (green) (n = 4). Scale bars: 100 μm. (F) Quantification of the numbers of CD31+ MHC-II + and CD4+ positive cells per visual field. (G) Immunofluorescent staining against IL-1, IL-6, and IL-17 and counterstaining with DAPI (blue: nuclei) in mouse control and ISO-model myocardial sections (n = 4). Scale bars: 100 μm. (H) Quantification of (G) on mouse myocardial sections from the control and ISO-induced myocardial fibrosis mice. All of the results are shown as the mean ± S.D.; ∗P < 0.05; ∗∗P < 0.01.

• 

  Fig. 5. ZL capsule improves ISO-induced cardiac dysfunction. (A) Representative images of echocardiography. (B) Echocardiographic assessment of EF%, FS%, LVESd, and LVEDd by quantification to evaluate the impaired cardiac function (n = 4). All of the results are shown as the mean ± S.D.; ∗P < 0.05; ∗∗P < 0.01.

• 

  Fig. 6. ZL capsule improved myocardial structure and reduced collagen deposition. (A, B) ZL capsule attenuates a series of symptoms after ISO-induced myocardial fibrosis. H&E, Sirius Red staining (Scale bar: 200 μm) and immunohistochemistry against α-SMA and immunofluorescence of collagen-I/III were performed and quantified in mouse myocardial sections (n = 4). Scale bar: 100 μm. All of the results are shown as the mean ± S.D.; ∗P < 0.05; ∗∗P < 0.01.

• 

  Fig. 7. ZL capsule can inhibit EndMT and the expression of MHC-II of EC. (A–C) Co-staining of sections from the ISO-induced myocardial fibrosis model for the EC marker CD31 and the mesenchymal markers α-SMA and FSP1, and quantification of the numbers of CD31+α-SMA+ and CD31+FSP1+ double-positive cells per visual field. (D–E) Co-staining of CD31 and the MHC class-II markers MHC-II, and quantification of the numbers of CD31+ MHC-II+ double-positive cells per visual field. Scale bar: 100 μm. All of the results are shown as the mean ± S.D.; ∗P < 0.05; ∗∗P < 0.01.

• 

  Fig. 8. ZL capsule inhibited the action of CD4+T cell and inflammatory reaction during myocardial fibrosis. (A) Flow cytometric analysis of the percentage of CD4+T cell in cardiac muscle tissues from the indicated experimental groups. (B, C) Immunofluorescent staining and quantification of CD4+ (red) T cell in mice after treatment with ZL capsule (n = 4). Scale bar: 100 μm. (D, E) Immunofluorescent staining and quantification of the number of IL-1+, IL-6+, and IL-17+ positive cells per visual field in mouse myocardium (n = 4). All of the results are shown as the mean ± S.D.; ∗P < 0.05; ∗∗P < 0.01.

• 

  Fig. 9. Schematic showing the complex mechanism of ZL capsule for reducing MF. Note 1. ZL capsule can inhibit the activation of myofibroblast cells by inhibiting EndMT, thereby reducing collagen deposition to improve myocardial fibrosis. Note 2. ZL capsule can attenuate the inflammatory response during fibrosis by intervening the activation of CD4+T cells mediated by EC.