Materials
The reagents and drugs used in this study are all commercialized, included Isoniazid, Rifampicin and Pyrazinamide (Shenyang Hongqi Pharmaceutical Co., Ltd.), siRNA oligos (Shanghai Jima Company), ALT and AST kits (Nanjing Jiancheng Institute of Biological Engineering), PrimeScript™ RT reagent Kit, SYBR® Premix Ex Taq™ II (TaKaRa, Japan), EZH2, Nrf2, NQO1 and HO-1 protein ELISA kits (Beijing Andy Huatai), and the ChIP-kit (Merck Millipore, Germany).
Animal model
Thirty-two 6-week-old SPF Kunming mice (Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.), half male and half female, weighing 18-22g, male and female segregation, cagewere raised in the SPF Animal Laboratory of the Animal Experiment Center, North China University of Science and Technology. After 1 week of adaptive feeding, the mice were randomly divided into 4 groups of 8 mice. The control group (Control) was given a solvent stomach gavage, and the model group (ADLI) was given isoniazid 90mg/Kg.d+rifampicin 135mg/Kg.d+pyrazine Amide 315mg/Kg.d mixed gavage for 4 consecutive days. Based on the ALDI model group, the inhibitor group (Si-EZH2) was injected with 200nmol/Kg EZH2-siRNA (CAA CAC CCA ACA CAU AUA ATT, UUA UAU GUG UUG GGU GUU GTT) into the tail vein while the negative control group (Si-Ctrl) was injected with 200nmol/Kg EZH2-NC-siRNA (UUC UCC GAA CGU GUC ACG UTT, ACG UGA CAC GUU CGG AGA ATT) based on the control group. On the 5th day after treatment, blood was collected and serum separated to detect ALT and AST levels using a chemical method. Mice were then sacrificed by cervical dislocation method and the liver tissue was taken. Part of the liver tissue specimen was fixed with 10% formaldehyde solution to observe pathological changes in the liver tissue.
RT-qPCR
The total RNA of liver tissue was extracted by the Trizol. Selecting RNA samples with the OD value detected by the microplate reader between 1.8 and 2.0. The total system of the reverse transcription system is 20 µl, of which Mix is 4 µl and the RNA mass is 1,000 ng, then the RNA volume is 1,000/RNA concentration, and the remaining system is supplemented to 20 µl with DEPC water. RT-qPCR was performed to detect EZH2, NRF2, NQO1, HO-1 mRNA levels using a standard protocol. The reaction system is 20 µl, Rox 0.4 µl, upstream and downstream primers 0.4 µl each, dH2O 6.8 µl, and cDNA 2 µl. The conditions are 95 °C for 30 s, 95 °C for 5 s, and 60 °C for 30 s, a total of 50 cycles. 2-ΔΔCt calculates the mRNA expression level. The data relative to the GAPDH gene expression levels. The primer sequences used are as follows: GAPDH (F: TTG GTA TCG TGG AAG GAC; R: TCA AAG GTG GAG GAG TGG), EZH2 (F: GGA CCA CAG TGT TAC CAG CAT; GTG GGG TCT TTA TCC GCT CAG), Nrf2 (F: CAG TGC TCC TAT GCG TGA A; R: TCT GGG CGG CGA CTT TAT), NQO1 (F: GAA GAC ATC ATT CAA CTA CGC C; R: GAG ATG ACT CGG AAG GAT ACT G), HO-1 (F: TCC TTG TAC CAT ATC TAC ACG G; R: GAG ACG CTT TAC ATA GTG CTG T).
ELISA assays and Western blotting
Mouse liver tissue was lysed using RIPA buffer containing PMSF to extract protein. An ELISA kit was used to detect EZH2, Nrf2, NQO1 and HO-1 protein levels according to the manufacturer’s instructions while Western blotting was used to detect EZH2 and Nrf2 proteins. The protein samples were separated by SDS-PAGE and transferred to PVDF membrane. The membrane was placed in a 5% skimmed milk powder shaker and sealed at room temperature. After being washed, the membrane was incubated overnight with EZH2 (1: 2,000) and Nrf2 (1: 2,000) primary antibodies 4. After the membrane was washed the next day, the secondary antibodies (1: 5,000) were incubated on the shaker at room temperature. They were developed and quantified by ECL color reagent in a gel imaging system.
ChIP assays
Chip assays were performed on mouse liver tissue using the ChIP-kit (Merck Millipore) according to the manufacturer’s instructions. Chromatin was cross-linked with 1% formaldehyde and immunoprecipitations performed with either anti-EZH2 or anti-H3K27me3 antibodies, and the level of H3K27me3 in the Nrf2 promoter region was determined by qPCR. The primer sequences used are as follows:F:5 '-CAG TGC TCC TAT GCG TGA-3', R:5 '-TCT GGG CGG CGA CTT TAT-3'.
Statistical analysis
Statistical analysis was performed with SPSS software (22.0; SPSS). In brief, the values are expressed as the mean ± standard deviation (SD). SNK test was used for comparisons between groups. P < 0.05 was considered statistically significant.