Amyloid β protein fragment 1 42

Salidroside (Macklin, S817419, purity > 98%), Dimethyl sulfoxide (DMSO, sigma-Aldrich, D2650), Ferrostatin-1(Fer-1, Topscience, 347174-05-4) was dissolved in DMSO and freshly diluted to the final concentration (0.05% DMSO) in the study, Glutamate(Glu, sigma-Aldrich, G1626), Amyloid β Protein Fragment 1–42(Aβ_1− 42, sigma-Aldrich, G1626), Cell Counting Kit-8 assay kit (CCK-8, bimake, B34304, USA), Lactate Dehydrogenase (LDH) detection kit (Best Bio, China, BB-4860-500T), Reactive Oxygen Species (ROS) assay kit (Nanjing jiancheng, E004-1-1), JC-1(Solarbio, J8030), C11 BODIPY 581/591(GLPBIO, 217075-36-0), FerroOrange Cell Ferrous Ion Fluorescence Probe (Dojindo, Japan, F374), Lipid Peroxidation MDA Assay Kit (Beyotime, S0131S), Total Superoxide Dismutase Assay Kit with WST-8 (Beyotime, S0101S), GSH and GSSG Assay Kit (Beyotime, S0053), LipoInsect™ Transfection Reagent (Beyotime, C0526-1.5mL), Opti-MEM™ I Reduced Serum Medium (Gibco, 31985070), Alexa Fluor 488-labeled Goat Anti-Rabbit IgG(H + L) (Beyotime, A0423), GPX4 Antibody (Affinity, DF6701), SLC7A11 Polyclonal Antibody (Proteintech, 26864-1-AP), HO1 Antibody (Affinity, AF5393), Nrf2 Polyclonal Antibody (Proteintech, 16396-1-AP), PCNA Polyclonal Antibody (Proteintech, 10205-2-AP), β-Actin (D6A8) Rabbit mAb (Cell Signaling Technology, #8457), HRP-conjugated Affinipure Goat Anti-Rabbit IgG(H + L) (Proteintech, SA00001-2).

All subjects were anesthetized with a ketamine/xylazine mixture (15 mg/kg + 1 mg/kg, ip) for i.c.v. stereotaxic surgery, which was performed in a standard rodent stereotaxic frame (David Kopf, USA).
The animals were divided into 4 groups of 8 each: the first group (VEH) was injected with 1 μl of isotonic saline solution (SS) followed by 5 μl of the same solution 30 min later; a second group (APN) was injected with 1 μl of adiponectin [150 ng/μl Adiponectin/Acrp (unconjugated) 1065-AP-050, Novus Biologicals] [65 (link)] followed by 5 μl of SS 30 min later; the third group (Aβ) was first injected with 1 μl SS and 30 min later with 5 μl of Aβ_1–42 (1 μg/μl, Amyloid β protein fragment 1–42, Sigma, A9810, dissolved in SS pre-incubated at 37 °C for 72 h in a shaking-water bath to induce aggregation); the last group (APN–Aβ) was administered with 1 μl of APN and 5 μl of Aβ_1–42. The stereotaxic coordinates employed for i.c.v. injection were measured as 0.92 mm posterior to bregma; 1.5 mm lateral to sagittal suture; 3.9 mm beneath the brain surface [80 (link)]. After surgery, animals were housed individually in an acrylic box cage (50 × 50 × 42 cm), and behavioral tests were performed 24, 48 and 72 h after the surgery as previously reported [54 (link)]. Animals that did not show alertness and proper motility were discarded from the study.

Application of Amyloid-β and FK506 was done as described before [28 (link)]. Briefly, Amyloid-β42 monomer (1 μM, Amyloid β-Protein Fragment 1–42, sigma) and/or FK506 (10 μM, Sigma) were applied into the ACSF after the 3 h recovery period and for 1 h before recording started.