Protective role of Tongxinluo in mitigating myocardial fibrosis in mice with acute myocardial infarction via neuregulin-1 upregulation and Inhibition of endothelium-interstitial transition

Fig. 1. Groups of mice were treated with different intervention. (A) Ligation site: left anterior descending branch of the left auricle 1–2 mm. AAV injection location was around the shaded area. (B) Sham group was only threaded without ligation, other groups were ligation of the LAD. NRG-1 OE group, NRG-1KD group and NC group were injected with the empty AAV vector. (C) After one week of adaptive feeding, the mice were modeled, then mice were sacrificed 4 weeks after modeling

Fig. 2. NRG-1 regulates cardiac function and cardiac morphology after AMI. (A) ECG: before ligation: sinus rhythm, normal ECG. After ligation: LAD, ST segment arched dorsal elevation. (B) After 4 weeks of modeling, the cardiac morphology in each group. (C) Cardiac hypertrophy index of each groups. (D) Echocardiography of each groups. Note: *P < 0.05 vs. Sham group, **P < 0.01 vs. Sham group; #P < 0.05 vs. Model group, ##P < 0.01 vs. Model group; &P < 0.05 vs. NRG-1 OE group, &&P < 0.01 vs. NRG-1 OE group

Fig. 3. Cardiac fibrosis after AMI, and regulation of NRG-1 can affect the degree of fibrosis. (A) HE staining in each groups. (B) and (C) Masson staining in each groups. (D) and (E). Immunohistochemical detection of Collagen I and Collagen III. Note: *P < 0.05 vs. Sham group, **P < 0.01 vs. Sham group; #P < 0.05 vs. Model group, ##P < 0.01 vs. Model group; &P < 0.05 vs. NRG-1 OE group, &&P < 0.01 vs. NRG-1 OE group

Fig. 4. Endothelial cells transform to mesenchymal cells after AMI, and regulation of NRG-1 can affect this transformation. (A) Immunohistochemical detection of endothelial cell marker CD31 and mesenchymal cell marker α-SMA in mouse heart. (B) Immunofluorescence CD31 (green), α-SMA (red) and DAPI (blue) stained apical images (original magnification, ×400, scale bar, 50 μm). (C) Western blot analysis to detect the protein expression levels of endothelial. (D) Western blot analysis to detect the protein expression levels of mesenchymal markers. (E) Western blot detects endothelial secretory function eNOs. Note: *P < 0.05 vs. Sham group, **P < 0.01 vs. Sham group; #P < 0.05 vs. Model group, ##P < 0.01 vs. Model group; &P < 0.05 vs. NRG-1 OE group, &&P < 0.01 vs. NRG-1 OE group

Fig. 5. Regulation of NRG-1 could affect downstream signaling pathway proteins. The expression of NRG-1, PI3K, p-AKT and AKT protein in different groups. Note: *P < 0.05 vs. Sham group, **P < 0.01 vs. Sham group; #P < 0.05 vs. Model group, ##P < 0.01 vs. Model group; &P < 0.05 vs. NRG-1 OE group, &&P < 0.01 vs. NRG-1 OE group

Fig. 6. TXL can improve the cardiac function of AMI mice, and knocking down the expression of NRG-1 can inhibit this effect. (A) Cardiac hypertrophy index of each groups; (B) Echocardiography of each groups. (C) Cardiac ejection fraction (EF), shortening fraction (FS), left ventricular end-systolic internal diameter (LVESD), and left ventricular end-diastolic internal diameter (LVEDD) in each group. Note: **P < 0.01 vs. Sham group; #P < 0.05 vs. Model group, ##P < 0.01 vs. Model group

Fig. 7. TXL can inhibit myocardial fibrosis in AMI mice, and knocking down the expression of NRG-1 can inhibit this protective effect. (A) Myocardial fibrosis of mice in each group (Masson staining)(×400). (B) Collage I and Collagen III expressions were detected by immunohistochemistry (×400). Note: **P < 0.01 vs. Sham group;#P < 0.05 vs. Model group, ##P < 0.01 vs. Model group; &P < 0.05 vs. TXL group. **P < 0.01 vs. Sham group; ##P < 0.01 vs. Model group; &P < 0.05 vs. TXL group

Fig. 8. TXL can inhibit endothelial mesenchymal transdifferentiation in AMI mice, and knocking down the expression of NRG-1 can inhibit this protective effect. (A) The expressions of CD31 and α-SMA were detected by immunofluorescence CD31 (green), α-SMA (red), DAPI (blue) (×400,50 μm). (B) The expression of CD31 and α-SMA protein in different groups. Note: *P < 0.05 vs. Sham group, **P < 0.01 vs. Sham group; #P < 0.05 vs. Model group, ##P < 0.01 vs. Model group; &P < 0.05 vs. TXL group

Fig. 9. Tongxinluo can inhibit the expression of NRG-1 and downstream pathway protein in AMI mice, and knocking down the expression of NRG-1 can inhibit this protective effect. The expression of NRG-1 PI3K and p-AKT protein in different groups. Note: *P < 0.05 vs. Sham group, **P < 0.01 vs. Sham group; #P < 0.05 vs. Model group, ##P < 0.01 vs. Model group