Chemicals and reagents
EMO (purity ≥ 97%), UDCA (purity ≥ 99%), SRT1720 (purity ≥ 98%, Sirt1 activator), EX527 (purity ≥ 98%, Sirt1 inhibitor), GW4064 (purity ≥ 98%, Fxr agonist), Z-Guggulsterone (GU, purity ≥ 98%, Fxr antagonist) and ANIT (purity ≥ 98%) were purchased from Macklin Biochemical (Shanghai, China). Alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TBIL) and total bile acid (TBA) assay kits were obtained from Nanjing Jiancheng Biotechnology (Nanjing, China). Antibodies for SIRT1, FXR, BSEP, MRP2, CYP7A1 and GAPDH were obtained from Affinity biosciences (Cincinnati, OH, USA). Antibodies for Horseradish peroxidase-labeled goat anti-rabbit IgG were obtained from Proteintech Group Inc. (Chicago, IL, USA). All the other chemicals of analytical grade were purchased from commercial sources.
Ethical approval
This investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication, revised 1985). All animal experiments were approved by Ethics Committee of The First Affiliated Hospital of Nanchang University (registration number: CDYFY-IACUC-202304QR046, date: 26-04-2023). The usage of samples and all methods were in strict accordance with the ARRIVE guidelines.
Animals and treatments
Male Sprague–Dawley (SD) rats (200±10 g) were obtained from Hunan SJA Laboratory Animal Co., Ltd (Changsha, Hunan, China, No. SYXK < Xiang > 2019-0017). They were housed under conditions of 55% humidity at 20-25°C, provided a standard diet with water available ad libitum and were kept on a 12 h light/dark cycle. The rats were randomly divided into the following 6 groups (n = 6): Control group, ANIT group, ANIT combined with UDCA (60 mg/kg) group and ANIT combined with EMO (20, 40, 80 mg/kg) group. Vehicle (0.2% CMC-NA) , UDCA (60 mg/kg) or EMO (20, 40, 80 mg/kg) alone were treated to rats by oral gavage once a day for 7 days. Rats in the control group were received olive oil and rats in other groups were given ANIT (75 mg/kg, dissolved in olive oil) intragastrically on day 57. After fasting for 12 h, all rats were sacrificed (pentobarbital sodium, 65 mg/kg, intraperitoneal injection) on day 7 to collect serum and liver tissue for further study.
Biochemical indexes and histological analysis
The levels of ALT, AST, TBIL and TBA were determined using commercial kits according to the manufacturer’s instructions. After rats were sacrificed, livers were collected, fixed in 4% formaldehyde, embedded in paraffin, sectioned at 5 µm, and stained with hematoxylin and eosin (H&E). Images were observed by Olympus BX43 light microscope (Tokyo, Japan).
L02 cells culture and treatments
The human normal hepatocyte cell line L02 was purchased from Fuxiang Biotechnology LLC (Shanghai, China). The L02 cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) and incubated at 37 ◦C with 5% CO2. Cells were seeded onto six-well plates at a density of 5 × 105 cells per well, and 24 h later, SRT1720 (5 μM), EX-527 (5 μM) , GW4064 (2 μM) or GU (2 μM) was added to the wells for 24 h. Then, the cells were treated with EMO (0.2, 0.6, 0.8 μM) or 0.1% dimethyl sulfoxide (DMSO) for 24 h. For the RNA silencing experiment, the cells were transfected with siRNA targeting Sirt1, or control siRNA using commercially available kits (Guangzhou RiboBio, Guangzhou, China).
Rat primary hepatocytes isolation, culture and treatments
Rat primary hepatocytes were prepared from male SD rats and seeded in collagen-coated plates as previously described7. The cells were cultured in serum-free Williams’ E medium. EX-527 (5 μM) or EX-527 (5 μM) + EMO (0.6 μM) were added to the wells for 24 h. Then, the cells were treated with ANIT (50 μM) or 0.1% DMSO for 24 h. The cells survival rate were determined by CCK8 (Dojindo Laboratories, Japan).
RT-qPCR and western blot analysis
Total RNA from liver and intestine was extracted using TRIzol Reagent (Invitrogen, CA, United States) according to the manufacturer’s instructions. Total RNA was reverse transcribed into cDNA using PrimeScriptTM RT Master Mix (Takara, Dalian, China), and then obtained cDNA was used as a template for real-time PCR amplification, performed by using SYBR Green (Takara, Dalian, China) and forward/reverse primer pairs for the tested genes. Independent reactions were performed in triplicate using an ABI StepOne Plus system (Applied Biosystems, CA, United States)12. Both forward and reverse primers used are presented in Table 1.
The total proteins were collected by lysis with RIPA lysis buffer. The nuclear proteins were prepared with nuclear and cytoplasmic protein preparation kit (APPLYGEN, Beijing, China). The total proteins were resolved on 10% SDS-PAGE gel and transferred to nitrocellulose membranes. The immunoreactive bands were detected using horseradish peroxidase-conjugated secondary antibodies and ECL reagents7. Specific protein bands were detected using enhanced chemiluminescence (ECL) kit (US EVERBRIGHT, Suzhou, China).
Statistical analysis
The data were analyzed by SPSS 25.0 software (Chicago, USA). The graphs were generated using GraphPad Prism 8.0 software (San Diego, USA). All data were presented as the mean±standard deviation (SD). The differences among the experimental groups were calculated by ANOVA, whereas student’s t-test was used to detect significant differences between two groups. p < 0.05 was considered statistically significant, and p < 0.01 was considered highly significant.