Gr assay kit

Cytosol and isolated mitochondria fractions were prepared in ice-cold lysis buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 1% IGEPAL, and 0.5% sodium deoxycholate) containing protease and phosphatases inhibitors cocktail (1861281, Thermo Fisher Scientific, Waltham, MA, USA). After incubation of 30 min on ice, samples were centrifuged at 15,000× g for 15 min at 4 °C. SOD activity was measured using a commercial SOD assay kit (ab65354, Abcam) and reading absorbance at 450 nm. Catalase activity was assessed by following the manufacturer’s protocol of catalase assay kit (707002, Cayman Chemical, Ann Arbor, MI, USA) and measuring absorbance at 540 nm. Then, glutathione reductase (GR) and glutathione peroxidase (GPx) activities were analyzed using GR assay kit (703202, Cayman Chemical) and GPx assay kit (ab102530, Abcam), respectively. Both enzyme activities were assessed at an optical density of 340 nm. SOD activity is expressed in U/mg of protein; catalase and GPx activities in µmol/min/mg of protein; and GR activity in µmol/min/mg of protein.

Neoponcirin, poncirin, and auraptene were purchased from ChemFaces (Wuhan, China). Umbelliferone, naringin, and imperatorin were obtained from Shanghai Sunny Biotech (Shanghai China), Sigma-Aldrich (St Louis, MO, USA), and ChromaDex (Irvine, CA, USA), respectively. The purity of the six reference standards was ≥95.0%. HPLC-grade methanol, acetonitrile, and water were obtained from J.T. Baker (Phillipsburg, NJ, USA). Glacial acetic acid, analytical reagent grade was purchased from Merck (Darmstadt, Germany). Other reagents were as follows: TP (T0028; Tokyo Chemical Ins. Co., Tokyo, Japan), corn oil (C8267; Sigma-Aldrich), (−)-riboflavin (Rib) (R9504; Sigma-Aldrich), Finasteride (Fin) (F1293; Sigma-Aldrich), testosterone enzyme-linked immunosorbent assay (ELISA) kit (582,701; Cayman Chemical, Ann Arbor, MI, USA), DHT ELISA kit (11-DHTHU-E01; ALPCO Diagnostics, Salem, NH, USA), catalase assay kit (707,002; Cayman Chemical), GPx assay kit (703,102; Cayman Chemical), GR assay kit (703,202; Cayman Chemical), SOD assay kit (706,002, Cayman Chemical), anti-proliferating cell nuclear antigen (PCNA) antibody (ab-29; Abcam, Cambridge, MA, USA) and anti-β-actin antibody (4967S; Cell Signaling Technology, Danvers, MA, USA).

After 24 h of treatment with the tested compounds, GR activity was measured using spectrophotometric GR Assay Kit (Item No. 703202, Cayman Chemical, Ann Arbor, USA). Cells were homogenized in cold buffer (50 mM potassium phosphate, pH 7.5, containing 1 mM EDTA) and centrifuged (10,000×g, 15 min, 4 °C), and the resulting supernatant (20 µl) was used to analyze enzyme activity and to determine protein concentration. GR catalyzes the reduction of oxidized glutathione (GSSG) to GSH with a consumption of one NADPH. Briefly, the reaction mixture contained 50 mM potassium phosphate (pH 7.5), 1 mM EDTA and 1 mM GSSG. The reaction was initiated by adding 50 µl NADPH and the decrease in absorbance caused by the oxidation of NADPH to NADP⁺ was measured once every 5 min at 340 nm. The results were presented as nmol/min/mg of protein.