Mmp 1

A total of 75,000 cells/mL were seeded and treated with ST09 (20, 40, 60, and 80 nM) for 48 h and the whole cell lysate was prepared as described [13 (link)]. Next, 30 µg of cell lysates was electrophoresed on 10 to 12% of SDS-PAGE (poly acrylamide gel electrophoresis) and were transferred to a polyvinylidene fluoride membrane (Millipore, Burlington, MA, United States). Blocking was performed using 5% skim milk in 1× PBS and then probed with primary antibodies: MMP2 from Biolegend, MMP1 from elabscience, Apaf, Bad, Bcl2, cytochrome c, Tubulin from Santa-Cruz Biotechnology, CA, and Caspase 9, Caspase 3, PARP, Vimentin, Bax, and GAPDH from Cell Signaling Technology, Beverly, MA, USA, followed by HRP-conjugated secondary anti-rabbit, anti-mouse antibodies (Cell Signaling Technology). The blots were developed using chemiluminescence reagent (Clarity Western ECL blotting substrate, Biorad) and the blot images were captured by the Chemidoc-XRS Biorad gel doc system. The protein band images were quantified using GelQuant.Net, BiochemLab solutions.

Sea Buckthorn proanthocyanidins (SBP, Purity: 91.5%) were provided by Puredia Limited (Qinghai, China). The SBP was extracted from Sea buckthorn by water extraction and macroporous resin column chromatography (MRCC), trademarked as CyanthOx™. HSFs (derived from human superficial skin tissue), fetal bovine serum (FBS), Dulbecco’s modified Eagle medium (DMEM), phosphate-buffered saline (PBS), penicillin–streptomycin solution (double antibody) 100×, and 0.25% trypsin solution were purchased from Xiamen Immocell Biotechnology Co., Ltd. (Xiamen, China). A Cell Counting Kit 8 (CCK-8) ROS detection kit was purchased from Beijing Solar Science & Technology Co., Ltd. (Beijing, China). A senescence β-galactosidase staining kit, SOD assay kit and WST-8, GSH assay kit, malondialdehyde (MDA) assay kit, and particular fixative solution, washing solution, blocking solution, Alexa Fluor 488-labeled Goat Anti-Rabbit IgG(H+L), and antifade mounting medium with DAPI for immunofluorescence staining were all purchased from Beyotime Inc. (Shanghai, China). Primary antibodies, including anti-TGF-β1, anti-Smad 3, anti-phospho-Smad 3 (Ser425), anti-Smad 4, anti-type I collagen, and β-actin, were obtained from Affinity Biosciences (OH, USA). MMP-1, MMP-3, and TIMP-1 Elisa assay kits were obtained from Elabscience Biotechnology Co., Ltd. (Wuhan, China).

MMP-1, MMP-3, MMP-13, Collagen II, IL-1β, TNF-α, IL-6, and iNOS were purchased from Elabscience, Hubei, China. NF-κB p65, COX-2, and PGE2 were purchased from Taiclone (Taipei, Taiwan). All analyses were performed according to the manufacturer’s instructions. Nitric oxide (NO) production was analyzed by the Griess reagent method. 100 μL of plasma and serially diluted standard (0.1 M Nitrite) were added to the 96-well plate. Then, 50 μL of SUL solution (0.1% sulfanilamide solution in 2.5% phosphoric acid), and 50 μL of NED solution (0.1% N-(1-naphthyl) ethylenediamine dehydrate chloride in dd water) were added. After 10 min, the absorbance was measured at 540 nm. The amount of NO was calculated by plotting the standard curve⁶⁶ .