Tgf β1

Proteins were extracted from cardiac fibroblasts or mouse myocardium by RIPA lysis buffer (Applygen, China) containing protease inhibitor cocktail (Sigma, Santa Clara, CA, USA). Total protein concentrations were measured by BCA assay (Pierce, USA). Proteins were then separated by SDS-PAGE and transferred to PVDF membranes (Merck Millipore, Germany). Membranes were incubated with primary antibodies collagen type I, Fibronectin (FN), Sec61α (Abcam, Cambridge, MA), TGF-β1, a-smooth muscle actin (α-SMA), connective tissue growth factor (CTGF) (Santa Cruz Biotechnology, CA, USA), α-Tubulin (Bioworld, USA), or Nogo-C (Abmart, China) and then incubated with secondary antibodies goat anti-rabbit IgG, goat anti-mouse IgG, rabbit anti-goat IgG (Biodragon, China), or mouse anti-rabbit IgG LCS (Abbkine, California, USA). Immunoblots were evaluated using the Chemi Doc XRS + instrument (Bio-RAD, Hercules, CA, USA).

Paraffin-embedded tissues were sectioned (5 µm), and antigen retrieval was performed using 10 mM sodium citrate buffer. Endogenous peroxidase activity was blocked by treating sections with 0.3% hydrogen peroxide in methanol for 15 min. Tissues were treated with polyclonal rabbit anti-transforming growth factor beta 1 (TGF-β1), anti-basic fibroblast growth factor (bFGF), and anti-platelet-endothelial cell adhesion molecule-1 (CD31) antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA; dilution 1:300) overnight at 4°C. Specific labelling was detected with a peroxidase-conjugated goat anti-rabbit IgG and avidin-biotin peroxidase complex. Slides were then mounted with coverslips and analyzed by two blinded pathologists. The expression of TGF-β1 and bFGF was assessed by immunoreactive cell density plus expression intensity. Immunoreactive cell density was graded semi-quantitatively as 1) less than 10% per field, 2) 10–30% per field, 3) 30–70% per field, or 4) more than 70% per field, whereas expression intensity was graded as 1) mild, 2) moderate, or 3) severe. At the same time, the number of microvessels was counted to assess CD31 expression. All assessments were performed in a blind manner by two investigators, and five regions of interest in each specimen were used for each determination.

Tissue samples taken from the wound area and 2 mm surrounding skin on days 0 and 7 after wound healing were fixed in formalin and embedded in paraffin. Frozen Sects. (4 μm thick) were cut, fixed in 4% paraformaldehyde for 15 min, then incubated with Triton X-100 for 10 min at room temperature. Sections were incubated with primary antibodies against EGR1 (1:100, Santa Cruz Biotechnology), TGF-β1 (1:150, Santa Cruz Biotechnology) or SOCS7 (1:100, Santa Cruz Biotechnology) at 4 °C overnight. The following day, samples were incubated with HRP-conjugated second antibodies for 30 min at room temperature, followed by the DAB Detection System kit (Servicebio, Wuhan, China). Cell nuclei were counterstained with hematoxylin. Samples were visualized using light microscopy.

Antibodies to p‐PDGFR‐β, p‐VEGFR2, p‐Src, Src, p‐STAT3, STAT3, p‐Smad3, Smad3, TGF‐β1, p‐NF‐κBp65, p‐Akt, Akt and β‐Actin were purchased from Cell Signaling Technology. TGF‐β1 and antibodies to type I collagen and fibronectin were purchased from Santa Cruz Biotechnology. p‐FGFR1 antibody was purchased from Life Span Biosciences. NF‐κBp65 antibody was purchased from Prosci Inc. Antibodies to CD68, CD31, MMP‐2, TIMP‐2, E‐cadherin, vimentin, Snail, Twist, and MCP‐1, TNF‐α, IL‐1β and IL‐6 ELISA assay kits were purchased from Abcam Inc. Nintedanib was purchased from Cayman. α‐SMA antibody, CG, Cell Counting Kit‐8 (CCK‐8) proliferation assay kit and all other chemicals were purchased from Sigma.