T akt

Chemical inhibitors were purchased from Calbiochem (LY294002, PD98059, SB203580), Sigma (SP600125), and SelleckBiochem (A66, TGX-221, CAL-101, AS-252424, Ruxolitinib). The doses used and specific targets for each inhibitor are summarized in Table 1. Recombinant proteins were purchased from Austral Biological (IGF-1) and R&D Systems (IL-1β, OSM, IL-6, sIL-6R). Antibodies were purchased from Cell Signaling Technology (p-Akt S473, T-Akt, p-ERK T202/Y204, T-ERK, p-JNK T183/Y185, T-JNK, p-p38 T180/Y182, T-p38), Santa Cruz Biotechnology (p-STAT3 Y705, T-STAT3), and Abcam (MMP-13, MMP-2).

Glucose and MK2206 were bought from Sigma-Aldrich (Sigma-Aldrich, Missouri, USA). Antibodiesagainst CD31 and decorin were bought from Boster (Boster, Wuhan, China), while antibodies against VEGF, p-AKT, T-AKT, p-c-JUN, T-c-jun and β-actin were purchased from Santa Cruz biotechnology (Santa Cruz, Texas, USA). Two antibodies against IGF1R were used. One was used for western blot and the other for neutralizing IGF1R. The former was a rabbit polyclonal antibody purchased from Boster that reacts with rat and human, as well as mouse, IGF1R. The latter antibody, used for neutralizing IGF1R, was purchased from Abcam (Abcam, Cambridge, UK) and blocks IGF1 by binding to its receptor. It is a mouse monoclonal antibody against the IGF1 receptor and mainly reacts with human IGF1R. The ECM gel was purchased from BD Bioscience (BD Bioscience, CA, USA). All other reagents were purchased from commercial suppliers unless otherwise indicated.

The cell or gastrocnemius muscle tissue was homogenized in a lysis buffer containing cOmplete™ protease inhibitor Cocktail (Roche Diagnostics, Indianapolis, IN, USA) and protease inhibitor (Sigma-Aldrich, St. Louis, MO, USA; Oakville, ON, Canada) and subsequently centrifuged. The BCA Protein Assay Kit (Bio-Rad, Hercules, CA, USA) was employed to determine the protein concentrations, ensuring an equal protein concentration across all experimental groups. The equivalent protein concentrations from each group were then subjected to SDS-PAGE. Following electrophoresis, the membranes were blocked with 5% skim milk and incubated with primary antibodies overnight. The primary antibodies utilized for Western blot analysis included IGF, p-AKT, t-AKT, p-mTOR, mTOR, p-PI3K, t-PI3K, MyoD, Myogenin, MuRF1, p-FOXO3a, FOXO3a, Sirt1, and PGC1α (Santa Cruz Biotechnology, Santa Cruz, CA, USA), as well as GAPDH and β-actin (Sigma-Aldrich, CA, USA) for use as a loading control. Subsequently, the membranes were incubated with the corresponding secondary antibodies to visualize the protein bands using an LAS3000 luminescent image analyzer (Fuji Film, Tokyo, Japan). β-actin served as the loading control, and Image J software (National Institute of Health, Bethesda, MD, USA) was employed for quantitative analysis.

HRMECs were counted and separated into three groups: a negative control; a high glucose damage model treated with safranal (10 μL, 80 g/mL); and a high glucose damage model treated with high glucose (10 μL, 25 mol/L). A Bio-Rad test kit was used to extract the cells’ total protein and evaluate the protein content (Bio-Rad, Hercules, CA, USA). Equal amounts of protein (30 μg) were resolved on polyacrylamide gels containing 12% Tris-HCl before being transferred to a PVDF blotting membrane (Millipore, Billerica, MA, USA). After blocking, specific antibodies against phosphate-extracellular regulated protein kinases (p-ERK), total-ERK (t-ERK), p-P38, t-P38, p-serine/threonine kinase (AKT), t-AKT (1:2000, Santa Cruz, CA, USA), E-cadherin, N-cadherin, Snail, Twist, Fibronectin(1:2000, Santa Cruz, CA, USA) and beta actin (1:2,000, Abcam, Cambridge, MA, USA), were treated with the membranes. The protein bands were detected by chemiluminescence after the blots had been carefully washed and treated with peroxidase-conjugated goat anti-rabbit or anti-mouse secondary antibodies (1:1,000, ZSGB-Bio, Beijing, China) (Pierce, Rockford, IL, USA). Three times the experiment was conducted (10 (link)).