1.Experimental Animals
Mice were placed in a temperature/humidity-controlled environment (24 ± 2°C/40%) and were maintained on a 12 h light/12 h dark cycle (8 a.m. and 8 p.m.) with free access to food and water. SIRT2 knockout mice were generated on a C57BL/6 background. All experimental procedures were approved by the Management Committee of Laboratory Animal Center of Heilongjiang Bayi Agricultural University. After 5 weeks of centralized feeding, the mice were divided into four groups, with 10 mice in each group. Inject CCl4 oil solution (50 ul/10g body weight, CCl4: olive oil = 1:9) into the abdominal cavity twice a week for 6 consecutive weeks. Before the end of the experiment, fast for 4 hours and measure blood biochemical indicators. After euthanasia, the liver of mice was taken for weighing, pathological analysis, and related protein analysis.
2.Serology tests
Serum levels of ALT、AST、TG、TC. were measured using Veterinary automatic biochemical analyzer (Seamaty, SMT-120VP).
3.Enzyme activity test
Commercial kits were utilized to determine the activity of endogenous antioxidant enzymes (CAT), superoxide dismutase (SOD), and glutathione (GSH) were obtained from Beyotime (Shanghai, China). MDA measurement was performed according to manufacturer's instructions using the 2-thiobarbituric acid (TBA) technique (Beyotime, Shanghai, China).
4.Coimmunoprecipitation (Co-IP)
Co-IP were performed as described by Song. Briefly, liver tissues and cells were lysed in IP-lysis buffer [50 mM Tris (pH 7.4), 150 mM NaCl, 0.1 mM EDTA, 20% glycerol, 0.2% NP-40, 0.1% SDS, protease and phosphatase inhibitors]. After freezing and centrifugation at high speed, the lysate supernatants were incubated with the indicated antibodies at 4℃. The pre-cleared protein A/G beads (Thermo Fisher Scientific; 88803) were then incubated with the supernatant at room temperature for 1.5 h. The collected proteins were analyzed by western blotting.
5.Histopathological analysis
Periodic Acid-Schiff, Sirius Red, Hematoxylin and eosin (H&E) and Masson staining were performed on serial sections (5µm thick) of liver tissue specimens. Then, the sections were observed under an optical microscope.
6.Immunofluorescence (IF) staining
For cell immunofluorescence staining, cells were grown on microscope cover glasses and treated as indicated. After treatment, the cells were fixed with 4% paraformaldehyde, blocked with 0.2% Triton X-100 for 15min and incubated with antibodies against Sirt2 (Proteintech, 19655-1-AP), NLRP3 (Cell Signaling Technology, 4053) overnight for approximately 12h under 4°C. The cells were then washed with PBS(Beyotime, C0221A) and incubated with fluorescently labeled secondary antibodies for 1 h under the room temperature, and 4ʹ,6-diamidino-2-phenylindole (DAPI) (Beyotime, C1005) was used to stain nucleus. The sections were observed under a confocal microscope (Nikon, Tokyo, Japan).
7.Immunohistochemical (IHC) Staining
The sections were stained with Collagen1 (Proteintech, 14695-1-AP), Collagen13 (Invitrogen, PA5-115039), TGF-β (Proteintech, 21898-1-AP) and α-SMA(Proteintech, 14395-1-AP) primary antibodies and later incubated with HRP polymer. A DAB Plus Substrate which added DAB Plus Chromogen was employed to visualize the staining.
8.Quantitative Real-Time PCR
Total RNA purification with Trizol (Invitrogen) and was treated retro-transcribed using the RT Kit (Takara, RR047A). According to the manufacturer’s instructions, quantitative real-time PCR experiments were performed. All reactions were repeatedly processed.
9.Western blot analysis
Western blot analysis was performed as described previously[ERS文章], Total protein lysates (30 µg) were immunoblotted with rabbit anti-Sirt2, rabbit anti-Collagen1, rabbit anti-Collagen13, rabbit anti-TGF-β, rabbit anti-α-SMA, rabbit anti-Keap1 (Proteintech, 60027-1-Ig), rabbit anti-Nrf2 (Proteintech, 80593-1-RR), rabbit anti-HO-1 (Proteintech, 10701-1-AP), rabbit anti-ASC (Proteintech, 10500-1-AP), rabbit anti-Caspase3 (Proteintech, 19677-1-AP), rabbit anti-Acetyl (Proteintech, 16087-1-AP), rabbit anti-NLRP3, mouse anti-β-actin (Proteintech, 66009-1-Ig), and then with the following secondary antibodies conjugated to horseradish peroxidase (HRP) for 1 h: HRP-conjugated Affinipure goat anti-mouse IgG(H + L) (Proteintech, SA00001-1) and HRP-conjugated Affinipure goat anti-mouse IgG(H + L)(Proteintech, SA00001-1).
10.ROS detection
ROS generation was detected by dihydroethidium (DHE) staining. Briefly, frozen liver sections were incubated with DHE (5 µmol/L) at 37°C for 30 min in a dark chamber. Fluorescent images were observed with a fluorescence microscope (Olympus IX53; Olympus, Tokyo, Japan).
11.Cell Culture and Treatment
LX2 and HEK293T were cultured in DMEM (Gibco, 11965092) supplemented with 10% fetal bovine serum and antibiotics at 37°C and 5% CO2 in a humidified incubator. LX-2 cells were treated with Ang II (Sigma, 4474-91-3) (10− 7 M) for 4 h.
12.Statistics
Statistical analyses were performed using GraphPad Prism 8.0 software (Chicago, IL, USA). Data are expressed as mean ± SD. Except where indicated, statistical significance was analyzed using two-tailed Student’s t-test and differences were considered statistically significant at P < 0.05