Isolation and Quantification of RNA

0.2 mL of Chloroform was added into Eppendorf containing PBMCs and 1 mL of TRIzol reagent (Thermo Fisher Scientific, Waltham, MA, USA), then centrifuged at 12,000×g for 15 min at 4 °C to remove debris. The supernatant was collected, placed in a new tube, mixed with isopropyl alcohol, and centrifuged at 12,000×g for 10 min at 4 °C. The supernatant was discarded, and the RNA pellet was washed twice with 1 ml of 75% ethanol. The pellet was air-dried for 30 min, dissolved with 30 μL of RNase-free water, and stored at − 80 °C after spectrophotometric quantification. About 500 ng of RNA was purified and subjected to reverse transcription using the cDNA with High-Capacity Reverse Transcription kit (Thermo Fisher Scientific, Waltham, MA, USA). Realtime PCR was performed on an Applied Biosystems^® 7500 QuantStudio 5 Real-Time PCR System (Life Technologies; Carlsbad, CA, USA). The qPCR analysis was conducted using a PowerUP SYBR green kit (Thermo Fisher Scientific, Waltham, MA, USA) and the following cycles: 95 °C for 2 min, followed by 95 °C for 15 s and 60 °C for 1 min for 40 cycles. The sequences of primers are reported in Table S3. The relative difference of NQO1, CAT and MT1E gene expression between Ops and CTRs was calculated using 2^-DDCT method and normalized to GAPDH levels as the internal control^{16 (link)}.