Rab0311

Blood was collected by cutting the tail tips of rats at 4 weeks after surgery (n = 5 for each group). The serum β-hydroxybutyrate (BHB) levels were determined using a metabolism assay kit (MAK041, Sigma-Aldrich). Then, transcardial perfusion was conducted with a heparinized-PBS solution. SN of these rats was quickly isolated and washed using ice-cold PBS. After being dried, weighed, grinded, homogenized, and centrifuged, the obtained supernatant was analyzed to determine the levels of tumor necrosis factor-α (TNF-α, RAB0480, Sigma-Aldrich), interleukin-1β (IL-1β, AB1832P, Sigma-Aldrich), and interleukin-6 (IL-6, RAB0311, Sigma-Aldrich) in SN through ELISA kits.

The obtained BAL fluid was centrifuged at 300 × g for 5 min (4˚C), and the resulting supernatant was collected for protein concentration analysis; the cell pellet was stored for further use. Briefly, 6–10 times the amount of red blood cell lysis buffer (Roche Diagnostics: 11814389001) was added to the cell pellet, and the cells were incubated on ice for 5 min. Ice-cold PBS was added to the cell lysate, which was then centrifuged at 500 × g for 5 min (4˚C). The supernatant was discarded, and the cells were centrifuged in 1 ml PBS (500 × g for 5 min, 4˚C) to remove any remaining red blood cell debris. The supernatant was discarded, and the cells were resuspended in 0.5 ml PBS and counted using a hemocytometer. The levels of inflammatory factors were determined using commercial ELISA kits (Sigma-Aldrich: T5944, RAB0311, SRP6511), and all experiments were performed according to the manufacturer's instructions.

Arterial blood was immediately placed in BD Vacutainer tubes (Becton Dickson, Franklin Lakes, NJ) after collection, mixed by hand, and kept at room temperature for 45 min, after which it was centrifuged (2000 g) at 4 °C for 20 min. Using a micropipetter, the serum was removed and immediately stored at − 80 °C for analysis. Interleukin (IL)-6 (RAB0311, Sigma, St. Louis, MO), IL-1β (RAB0277, Sigma), and tumor necrosis factor (TNF)-α (ab100785, Abcam, Cambridge, UK) were measured according to the manufacturer’s instructions. For all assays, both the standards and unknowns were measured in duplicate.