Ss 31

Mice were randomly assigned to one of the following four groups (n = 18 each): control, control + SS-31, isoflurane, and isoflurane + SS-31. SS-31 (5 mg/kg, China Peptides Co, Ltd, Shanghai, China) or phosphate-buffered saline (PBS; vehicle) was intraperitoneally administered to the mice with a volume of 0.4 mL/kg 30 min before gas inhalation. The dose of SS-31 was chosen based on previous optimization in mouse models [15 –18 (link)]. Anesthesia was induced by placing the mice in an anesthetizing chamber prefilled with 1.8% isoflurane plus 30% oxygen (O₂) for 10 min and then changed to 1.5% isoflurane for 110 min. For control experiments, 30% O₂ was delivered for 2 h at the same flow rate. The composition of the chamber gas was continuously monitored using a Datex^TM infrared analyzer (Capnomac, Helsinki, Finland). Mice were kept normothermic throughout the experiment.
Two experiments were performed. In experiment one, six of ten mice in each group were immediately decapitated 2 h after gas inhalation, and the hippocampus was rapidly removed and separated into two halves for mitochondria isolation and biochemical assays, respectively. The other four mice in each group were perfused for immunohistochemical analyses. In experiment two, eight mice in each group were used for behavioral tests 24 h after the 2 h-gas inhalation.

SS-31 (synthesized by China Peptides Co, Ltd, Shanghai, China) or normal saline as a vehicle was administered to mice by subcutaneous injection for 12 weeks. Mice were randomly assigned to one of the following three groups (n = 15 each): Placebo (P, saline administration), SS-31 administration with dose of 1 mg/kg/d (M1) and 3 mg/kg/d (M3). The doses of SS-31 were chosen based on previous optimization in mouse models. The injection time was between 9 and 10 AM. No mice were dropped out during the experiments.

Bafilomycin A1, chloroquine, N-acetyl-L-cysteine (NAC), and MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine hydrochloride) were purchased from Sigma-Aldrich; etoposide was purchased from Selleck. SS-31 was purchased from China Peptides. Ammonium chloride (NH₄Cl) was purchased from Enox. Hydrogen peroxide was purchased from Shanghai Experiment Reagent Company. Anti-HA-Tag mouse monoclonal antibodies (HRP conjugate), -PARP (46D11) rabbit monoclonal antibodies, -caspase-3 rabbit polyclonal antibodies, -cathepsin D rabbit polyclonal antibodies, and -histone 3 (D1H2) rabbit monoclonal antibodies were purchased from Cell Signaling Technology. An anti-α-tubulin (ab80779) mouse monoclonal antibody and TFEB antibodies (ab113372) were purchased from Abcam. Anti-C-ETS2 (c-20) rabbit polyclonal antibodies were purchased from Santa Cruz Biotechnology, -LMNB1 (A2452) antibody was purchased from ABclonal, and -β-actin mouse monoclonal antibodies were purchased from Sigma-Aldrich. For immunohistochemical purposes, we used anti-tyrosine hydroxylase antibodies (T2928, Sigma) and -rabbit GFP antibodies (number 2956, Cell Signaling Technology).

Male C57BLKS/J db/db diabetic (n = 12) and littermates of non-diabetic heterozygous db/m (n = 6) mice aged 8 weeks were purchased from the Model Animal Research Center of Changzhou Cavens (Jiangsu, China). The animals were housed in Shanxi Medical University with a temperature of 21–25 ℃, humidity of 50–60%, a 12 h:12 h light–dark cycle, and free access to a regular diet and pure drinking water. Half of the db/db mice (n = 6) were injected intraperitoneally with 3 mg/kg SS-31 (D-Arg-2′6′-dimethylTyr-Lys-Phe-NH2; ChinaPeptides, Shanghai, China) daily for 10 weeks. The dosage (3 mg/kg/day) was based on related studies showing the efficacy of SS31 without adverse effects [28 (link)]. The db/m mice and the other half of the db/db mice (n = 6) were injected with a 0.1 mol/L sodium citrate solution. The mice were euthanized at 18 weeks old, and serum, urine, and kidneys were harvested for further biochemical and histological analysis. All experimental protocols were conducted according to the Ethics Review Committee for Animal Experimentation of Shanxi Medical University.

Human healthy control and FRDA patient-derived lymphoblasts and fibroblasts were purchased from the Coriell Institute for Medical Research repository. The cell lines used: GM14519, GM15849, GM15850, MCH46, GM03816, GM04078. Cells were cultured in medium RPMI 1640 (Solarbio Science & Technology Co. Ltd., Beijing, China) with 10% FBS, penicillin (100 U/mL) and streptomycin (100 mg/mL) at 37 °C in a 5% CO₂ tissue culture incubator. SS-31 (synthesised by ChinaPeptides Co., Ltd, Shanghai, China) was dissolved with PBS and stored in −80 °C.

Unless stated otherwise, all reagents were purchased from Sigma Aldrich. MitoQ was acquired from MedKoo Biosciences and SS‐31 from China Peptides.