**4. Materials and Methods**

#### *4.1. Isolation of Serum Exosomes*

Whole blood was collected from young (three-month-old) or old (22-month-old) rats (*n* = 6 per group) via cardiac puncture into BD Vacutainer®Plus Glass Serum blood collection tubes (Becton Dickinson, NJ, USA). Whole blood samples were allowed to clot by standing at room temperature for 30 min, and the clots were removed by centrifugation for 10 min at 1000× *g* at 4 ◦C. The isolated serum samples were aliquoted and stored at −80 ◦C.

Serum exosomes from young or old rats were isolated using the ExoQuick Exosome precipitation kit (System Biosciences, CA, USA). Briefly, serum (500 μL) was centrifuged at 3000× *g* for 15 min at 4 ◦C to eliminate cells and cell debris. The supernatant was transferred to a sterile micro-tube, and an appropriate volume of exosome precipitation solution from the kit was added, with incubation for 30 min at 4 ◦C. The mixture was then centrifuged at 1500× *g* for 30 min at 4 ◦C, and the exosome pellet was re-suspended in sterile phosphate-buffered saline at 4 ◦C.
