Mmp 1

We extracted blood samples from the heart of the test mice on the final day of experiments. The plasma levels of MMP-1, MMP-2, MMP-9, histamine, hyaluronidase, hyaluronic acid, IL-6, TNF-α, and AGEs were determined using commercial ELISA kits (MMP-1: MyBioSource, San Diego, CA, USA; MMP-2, MMP-9, hyaluronic acid, and TNF-α: R&D Systems, Minneapolis, MN, USA; histamine: Bertin Pharm., Montigny-le-Bretonneux, France; hyaluronidase: AB Clonal Inc., Tokyo, Japan; IL-6: Proteintech, Rosemont, IL, USA; and AGEs: OxiSelect AGE connection ELISA kit, Cell Biolabs Inc., San Diego, CA, USA), according to the instructions of the respective manufacturers.

Dorsal skin tissue samples were homogenized in PBS and centrifuged at 5000 rpm for 10 min. The supernatants were collected and used for enzyme-linked immunosorbent assay (ELISA). The protein content of the supernatant was assayed using a bicinchoninic acid (BCA) protein assay kit (Thermo Scientific, Rockford, IL, USA). The content of hyaluronic acid (R&D Systems, Minneapolis, MN, USA), collagen (Abcam, Cambridge, UK), MMP-1 (MyBioSource, San Diego, CA, USA), and MMP-9 (R&D Systems, Minneapolis, MN, USA) in skin homogenates was measured using the ELISA kits, according to the manufacturer’s instructions.

Blood was collected from the hearts of mice at the determined end points and centrifuged at 3000 rpm to isolate the plasma by a microvolume high-speed cooling centrifuge MX-201 (TOMY, Tokyo, Japan) and stored at −20 °C. The dorsal skin, livers, and kidneys were excised and homogenized with lysis buffer (Kurabo, Osaka, Japan), and then, the tissue lysate was centrifuged at 10,000 rpm. Plasma concentrations of interleukin (IL)-6, tumor necrosis factor (TNF)-α, creatinine, hyaluronic acid, endostatine, glutamate oxaloacetate transaminase (GOT), glutamic acid pyruvate transaminase (GPT), matrix metalloproteinase (MMP)-1, histamine, and angiopoitin 1 and 2 were measured using appropriate ELISA kits according to manufacturers’ instructions (IL-6, Enzo Life Sciences, Farmingdale, NY, USA; TNF-α, hyaluronic acid, and angiopoietin 2, R&D Systems, Minneapolis, MN, USA; MMP-1, MyBioSource, San Diego, CA, USA; histamine, Bertin Pharm, Montigny-le-Bretonneux, France; creatinine, Cayman, Ann Arbor, MI, USA; Endostatine, BioMedica, Vienna, Austria; angiopoietin 1, EIAAB Science, Wuhan, China; GOT and GPT, Wako, Osaka, Japan; ROS, Cell Biolabs Inc., San Diego, CA, USA). Optical density was measured using a microplate reader (Molecular Devices, Sunnyvale, CA, USA).