Gpx cellular activity kit

The glutathione peroxidase (GPx) cellular activity kit (from Sigma chemicals) was used to evaluate the GPx. This kit is based on an indirect method, consisting of the oxidation of glutathione (GSH) to oxidized glutathione (GSSG) catalyzed by GPx, which is then coupled with recycling GSSG back to GSH utilizing glutathione reductase (GR) and NADPH. The absorbance of the solutions was recorded at 340 nm. Thus, a decrease in NADPH during the oxidation to NADP is indicative of GPx activity [40 (link)].

An analysis of the expression of proteins associated with SIRT1/Nrf2/HO-1 axis was conducted. The hippocampal content of SIRT1 was quantified by AFG Bioscience ELISA kit (Cat. # EK720561; AFG Bioscience, Northbrook, IL, USA). Regarding hippocampal levels of Nrf2, Cayman nuclear extraction kit was used for the isolation of total proteins in the nuclear compartment as instructed by the provider (Cat. # MBS012148; Cayman Chemical, Ann Arbor, MA, USA). Then, Nrf2 protein levels in the nuclear compartment were determined by a commercial ELISA kit (Cat. # EK720003; AFG Bioscience, Northbrook, IL, USA). The protein expression levels of HO-1 were determined by an ELISA kit purchased from Elabscience (Cat. # E-EL-R0488; Elabscience, Wuhan, China). For SIRT1, Nrf2, and HO-1, the final color reading was taken at a wavelength of 450 nm. Regarding glutathione peroxidase (GPx) activity, the enzymatic activity measurement was applied using Sigma-Aldrich GPx cellular activity kit under the guidance of the vendor (Cat. # CGP1; Sigma-Aldrich, St. Louis, MO, USA). To this end, absorbance decline at 340 nm was tracked by a kinetic program. Quantification of the lipid peroxides, a well-characterized oxidative stress marker, was performed as previously reported by Buege and Aust [54 (link)]. The final color reading was taken at a wavelength of 535 nm.