Specific primers

Txnrd1, Txnrd3, Txn1, Txn2, Nrf2, keap1, and p65 transcript expression levels in the liver tissue were detected by real-time fluorescent quantitative PCR (Applied Biosystems, Foster City, CA, USA). RNA was extracted from liver tissue according to the manufacturer’s instructions, and the TAKARA reverse transcription kit-RR047A was used to obtain complementary DNA (cDNA). The reverse-transcribed cDNA was used as a template, β-actin acted as an internal reference gene, and specific primers (Sangon Biotech, Shanghai, China) of the target genes were synthesised according to the sequences in Table 1. A fluorescence quantitative PCR amplification system (cDNA template: 1 μL, Primer F: 0.5 μL, Primer R: 0.5 μL, TB Green: 7.6 μL, Rox: 0.4 μL (Takara Biomedical Technology Co., Ltd, Beijing, China), ddH2O: 10 μL). The PCR conditions were as follows: 5 min at 95 °C, 40 cycles of 10 s at 95 °C, 30 s at 60 °C, and 30 s at 72 °C followed by a melting curve step. The mRNA expression level was calculated using the 2^−△△CT method and the data were analysed.

Total RNA was extracted by RNAiso Plus (Takara, Dalian, China). The reaction mixture (20 μl) containing 1 μg of total RNA was reversely transcribed to cDNA by using PrimeScript RT-polymerase (Takara, Dalian, China). Quantitative PCR was performed on the cDNA using specific primers (Sangon, Shanghai, China) for HULC (F:5‘-TCATGATGGAATTGGAGCCTT-3′, R: 5′-CTCTTCCTGGCTTGCAGATTG-3′), miR-200a-3p (5′- AACACTGTCTGGTAACGATGTCGT-3′), ZEB1 (F: 5′- CAGCTTGATACCTGTGAATGGG-3′, R: 5′-TATCTGTGGTGTGGGACT-3′) and GAPDH (F: 5′-CAGCCAGGAGAAATCAAACAG-3′, R: 5′-GACTGAGTACCTGAACCGGC-3′). All reactions were performed using the Applied Biosystems 7000 Sequence Detection System (Applied Biosystems, Foster City, CA, USA). Relative expression levels were calculated as ratios normalized against those of GAPDH. Comparative quantification was determined using the 2^{−Δ ΔCt} method.

Total RNA of the hippocampus was isolated using a Eastep® Super RNA extraction kit (Promega, Shanghai, China) followed by reverse transcription with a GoScript™ Reverse Transcription Kit (Promega, Shanghai, China) according to the manufacturer’s protocol. Finally, RT-PCR was performed with GoTaq® qPCR Master Mix (Promega, Shanghai, China) and specific primers (Sangon Biotech, Shanghai, China). The relative fold change in gene expression was calculated with the 2^−ΔΔCt method with GAPDH as the internal control [34 (link)]. The primers used to detect TNF, IL-6, IL-1β, KCC2, and GAPDH mRNA were as follows:

TNF forward: 5’-CTGTGAAGGGAATGGGTGTT-3’;

TNF reverse: 5’-CAGGGAAGAATCTGGAAAGGTC-3’;

IL-6 forward: 5’-GGCCCTTGCTTTCTCTTCG-3’;

IL-6 reverse: 5’-ATAATAAAGTTTTGATTATGT-3’;

IL-1β forward: 5’-AGTTGACGGACCCAAAAG-3’;

IL-1β reverse: 5’-AGCTGGATGCTCTCATCAGG-3’;

KCC2 forward: 5’- AGGTGGAAGTCGTGGAGATG-3’;

KCC2 reverse: 5’-CGAGTGTTGGCTGGATTCTT-3’;

GAPDH forward: 5’-GACATGCCGCCTGGAGAAAC-3’;

GAPDH reverse: 5’-AGCCCAGGATGCCCTTTAGT-3’.