Tnf α

Rabbit antibodies, such as TOM20 (WL0706), BAX (WL01637), BCL-2 (WL01556), cytochrome c (WL02410), caspase 3 (WL04004), iNOS (WL0992a), VDAC (WL02790), IL-2 (WL03259), TNF-α (WL01581), IL-1β (WLH3903), and β-actin (WL01372), were procured from Wanleibio in Shenyang, China, while rabbit anti-MFF (AF2365), anti-Mid49 (DF12044), anti-Mid51 (DF12019), anti-Opa1 (DF8587), and anti-Mfn1 (DF7543) were obtained from AFFINITY, Co., Ltd. This study acquired goat anti-rabbit IgG/HRP (PAB21463HRP-1000) secondary antibody (1 : 10000) from EarthOx (Millbrae, USA). The reagents and chemicals were all products of Sigma (St. Louis, USA). We procured SH-SY5Y cells from the Type Culture Collection of Chinese Academy of Sciences (Shanghai, People's Republic of China) and cultured them using DMEM/F-12 involving fetal bovine serum (FBS, 10%) and PS (penicillin–streptomycin, 1%). Meanwhile, BV2 microglial cells, which were purchased from the iCell Bioscience Inc., Shanghai, were cultured using DMEM (HyClone, UT, USA) involving FBS (10%; Gibco, CA, USA) and PS (1%). Male SD (Sprague–Dawley) rats, whose weights ranged from 250 to 280 g, were offered by Jinzhou Medical University. The present experimental protocol conformed to the National Guidelines for Animal Protection and gained approval from foregoing university's Institutional Animal Care and Use Committee.

The enzyme‐linked immunosorbent assay (ELISA) kits were used to detect the expression level of IL‐6 (Wanleibio, China), IL‐1β (Wanleibio, Shenyang, China), TNF‐α (Wanleibio, Shenyang, China), Lactic dehydrogenase (LDH; Wanleibio, Shenyang, China), and total protein (TP; Nanjing Jiancheng Bioengineering Institute, China) in mice serum according to the manufacturer instructions.

LP was procured from Beijing Tongrentang Technology Development Co. (Beijing, China). D-Gal, >99% pure, was purchased from Sigma-Aldrich (St. Louis, MO, USA). The antibody to p-AMPK was purchased from Cell Signaling Technology (Boston, USA), and the antibody to SIRT1 was ordered from Affinity Biosciences (USA). The antibody to NF-κB (P65) was bought from BOSTER (Wuhan, China). The antibodies to AMPK, HO-1, IL-1, IL-6, and IL-10 were all purchased from Boiss (Beijing, China), and the antibodies to P21^Waf1/Cip1, P16^INK4A, and TNF-α were procured from Wanleibio (Shenyang, China). HRP-conjugated secondary antibodies were purchased from Boiss. The chemiluminescence reagents were from Affinity Biosciences (USA).

The liver tissues were lysed with Radio Immunoprecipitation Assay (RIPA) buffer (Beyotime, P0013B, Shanghai, China) or Nuclear Extraction Kits (Beyotime, P0028, Shanghai, China) supplemented with Protease Inhibitors (Beyotime, P1011, Shanghai, China). Then, Western blot was carried out as the general process. Primary antibodies: NF-κB (Cell Signaling Technology, 8242S, 1:1000, American), TNF-α (Wanleibio, WL01581, 1:1000), Nrf2 (Wanleibio, WL02135, 1:500, Shenyang, China), HO-1 (Wanleibio, WL02400, 1:500, Shenyang, China), Caspase-3 (Santa Cruz Biotechnology, sc-56053, 1:500, American), Bax (Cell Signaling Technology, 2772S, 1:1000, American), β-actin (Cell Signaling Technology, 4970S, 1:1000, American), and H3 (Proteintech, 17168-1-AP, 1:1000, American). Secondary antibodies: HRP-labeled goat anti-rabbit IgG(H + L) (Beyotime, A0208, Shanghai, China) and HRP-labeled goat anti-mouse IgG(H + L) (Beyotime, A0216, Shanghai, China).

RIPA buffer containing protease inhibitors (Beyotime) was used to lyse tissues. The primary antibodies were as follows: IL-6 (Wanleibio, Shenyang, China,), TNF-α (Wanleibio), Caspase-3 (Cell Signaling Technology, Danvers, MA, USA), iNOS (Santa Cruz Biotechnology, Santa Cruz, CA, USA), Bax (Cell Signaling Technology), SIRT 1 (Wanleibio), p53 (Wanleibio), p21 (Wanleibio), SA-β-gal (Cell Signaling Technology), p16 (Wanleibio), Nrf2 (Beyotime), HO-1 (Wanleibio), and β-actin (Wanleibio). The secondary antibodies were as follows: HRP-labeled goat anti-mouse and anti-rabbit IgG(H + L) (Beyotime).