Sirt1

Total proteins from tissues and cells were harvested, washed, and lysed in radioimmunoprecipitation buffer (RIPA) buffer. The protein concentration of each purified exosome sample was determined using a bicinchoninic acid (BCA) protein assay kit (CWBIO). Equal amounts of tissue or cell lysates were separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene fluoride (PVDF) membranes. After being blocked with 5% skim milk for 1 h, the membranes were incubated with primary antibodies and horseradish peroxidase (HRP)-conjugated secondary antibodies and detected using an enhanced chemiluminescent (ECL) substrate detection system. The primary antibodies used in the experiments were as follows: CD9 (1:500, Bioworld Technology, USA), CD63 (1:500, Bioworld Technology, USA), Bax (1:500, Bioworld Technology, USA), Bcl2 (1:500, Bioworld Technology, USA), SIRT1 (1:500, Bioworld Technology, USA), PCNA (1:500, CST, USA), 14-3-3ζ (1:500, Bioworld Technology, USA), TNF-α (1:500, Bioworld Technology, USA), Cleaved caspase3 (1:400, Bioworld, USA) GAPDH (1:2000, CWBIO, China), β-actin (1:2000, CWBIO, China). Primary antibodies were incubated overnight at 4° C. The HRP-conjugated goat anti-rabbit and goat anti-mouse secondary antibodies (1:2000, CWBIO, China) were incubated 2 hours at 37° C.

DMEM and fetal bovine serum (FBS) were purchased from Life Technologies (Grand Island, NY, United States). PA used for cell treatment was purchased from National Institutes for Food and Drug Control (Guangzhou, China). PA used in animal trial was from Zelang (purity > 98.0%, HPLC; Nanjing, China). Metformin Hydrochloride Tablets used in animal experiments were purchased from Bristol-Myers Squibb Company (Shanghai, China). DMSO and STZ were purchased from Sigma-Aldrich (St. Louis, MO, United States). Primary antibodies against FN (catalog: sc-18825) and ICAM-1 (catalog: sc-1511) were from Santa Cruz Biotechnology (Dallas, TX, United States); Antibodies against Nrf2 (catalog: 16396-1-AP) and SOD-1 (catalog: 10269-1-AP) were purchased from Proteintech Group (Wuhan, China); Sirt1 (catalog: BS6494) was from Bioworld Technology (St. Paul, MN, United States); HO-1 (catalog: A1346) was from Abclonal Technology (Baltimore Avenue, United States); Lamin B1 (catalog: ab-133741) was from Abcam (Cambridge, MA, United States); Anti-rabbit and anti-goat secondary antibodies were purchased from Beyotime (Haimen, China); Alexa Fluor^® 488 goat anti-rabbit IgG (catalog: A-11008) was purchased from Molecular Probes (Eugene, OR, United States).

After adding 200 μL of the lysate to the 6-well plate, it was allowed to stand for 20 minutes on ice, and the liquid was collected and centrifuged (13,000 rpm, 15 min); then, the supernatant was collected. The concentration of the protein was determined by the bicinchoninic acid (BCA) (Camilo Biological, Nanjing, China) method and quantified. The protein was separated using a 10% sodium dodecyl sulfate-polyacrylamide gel, then transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, Billerica, MA, USA) for 2 hours at 4°C. 5% skim milk was prepared with Tris-buffered saline with Tween-20 (TBST) to block the specific antigen for 2 hours. After washing with TBST for 1 minute, PVDF membranes were incubated with a specific primary antibody (SOD1, Abcam, Rabbit; 1 : 3000; SOD2, Abcam, Rabbit, 1 : 3000; GPX1, US 1 : 1000, CST; GPX3, US 1 : 1000, CST; Bcl-2, Abcam, Rabbit, 1 : 2000; Bax, Abcam, Rabbit, 1 : 500; Sirt1, Bioworld, mouse, 1 : 500, China; P53, Abcam, Rabbit, 1 : 2000; ROCK1, Abcam, Rabbit, 1 : 500; ROCK2, Abcam, Rabbit, 1 : 1000; MYPT-1, Abcam, Rabbit, 1 : 1000; GAPDH US 1 : 1000 CST) at 4°C overnight. The next day, TBST was washed for 30 minutes. Specific proteins were detected by secondary antibodies and observed by the electrochemiluminescence (ECL) (Pierce, Rockford, IL, USA) system.