Clinical Samples
Clinical skin samples from patients with psoriasis and healthy persons (same age and sex as patients) were collected from Huashan Hospital (Shanghai, China). The experiment was approved by the Huashan Hospital Clinical Research Ethics Committee. All methods were carried out in accordance with relevant guidelines and regulations, and all participants/donors provided written informed consent for the study.
Animal Experiments
All male BALB/c mice (10 weeks old, 25-35 g) were obtained from the Animal Experiment Centre of Huashan Hospital and fed under sterile specific-pathogen-free (SPF) conditions. The mice were randomly assigned into four groups (n=3 each) for further study, and the mice in group A were not treated. In groups B-D, mice were evenly smeared with 62.5 mg imiquimod cream on their backs once a day for three consecutive days. On the fourth day, 50 mg imiquimod cream was applied evenly on the backs of the mice once a day for two consecutive days. On day seven, the psoriasis animal models (imiquimd-induced mouse model of psoriasis, IMD) were successfully established in the B-D groups. In group B, normal saline was intragastrical injected for the positive control group (model group). MTX has been used to treat psoriasis and other skin diseases for more than 50 years [13]. For mice in group C, 20 mg/kg MTX was intragastrical injected for the treatment control group (model + MTX group). In addition, in mice of group D, 20 mg/kg glycyrrhizin was intragastrical injected for the treatment observation group (model + glycyrrhizin group). In groups B–D, the treatment was performed once a day for four consecutive days, and on the eleventh day, the back skin of the mice in groups A-D was photographed, and the mice were sacrificed by cervical dislocation. The animal experiments used meet the requirements of animal welfare and animal ethics of Huashan Hospital, and are approved by the Ethics Committee of animal experiments of Huashan Hospital.
Cell culture and treatment
HaCaT cells were purchased from the cell bank of the Chinese Academy of Sciences and met the cell line STR identification criteria. HaCaT cells were cultured in Dulbecco's Modified Eagle’s Medium (L110, Basamedia Biology, Shanghai, China) supplemented with 10% fetal bovine serum (Thermo Fisher), 100 g/mL penicillin, and 100 g/mL streptomycin (L110, Basamedia Biology, Shanghai, China) in a moist incubator with 5% CO2 at 37 °C. Wild type HaCaT cells were treated with IL-17A (100 ng/mL), glycyrrhizin (2 μM), and IL-17A (100 ng/mL) + glycyrrhizin (2 μM) for 48 h. The IL-17A-HaCaT cells (treated with 100 ng/mL IL-17A) were treated with glycyrrhizin (2 μM) and EX527 (100 nM, SIRT1 inhibitor, Med Chem Express) for 48 h.
Cell-counting kit-8 (CCK-8)
HaCaT cells were seeded in a 96-well plate at a density of 5×103 cells per well (triple replication). The next day, after the HaCaT cells adhered, the culture solution was discarded, and a mixture of 10 µL CCK-8 solution + 90 µL serum-free medium was added, and the cells were cultured in an incubator for 2 h. After 2 h, the absorbance of the cells at 450 nm was measured using a microplate reader (SpectraMax M2e; Molecular Devices, Sunnyvale, CA, USA). The above steps were repeated for five consecutive days.
Immunohistochemical experiments
For the immunohistochemical analyses, skin samples were dewaxed and rehydrated, followed by endogenous peroxidase quenching, antigen retrieval (saline sodium citrate, autoclaving), and blocking with 10% goat serum. The sections were then incubated with anti-rabbit STAT3 antibody (1:2000, CST, USA), phosphorylated STAT3 (p-STAT3) antibody (1:2000, CST, USA), acetylated STAT3 (a-STAT3) antibody (1:2000, CST, USA), SIRT1 antibody (1:2000, CST, USA), AMPK antibody (1:2000, CST, USA), phosphorylated AMPK (p-AMPK) antibody(1:2000, CST, USA), and α-tublin antibody (1:2000, CST, USA). After washing with PBS, the sections were incubated for 50 min with secondary antibody, stained with DAB working solution, and then counterstained with hematoxylin.
Enzyme linked immunosorbent assay (ELISA)
The cell lysis solution was collected to detect the expression of IL-6 (1:2000, Abcam, USA), TNF-α (1:2000, Abcam, USA), and CCL20 (1:2000, Abcam, USA) according to the manufacturer’ s instructions. The absorbance was then measured with a microplate reader (SpectraMax M2e, USA) at 450 nm.
Real-time fluorescence quantitative PCR (RT-PCR)
Real-time fluorescence quantitative PCR (RT-PCR)
Trizol reagent (Thermo Fisher Scientific) was used to extract total RNA from cells and tissues, and a reverse transcription kit (TaKaRa) was used to reverse the total RNA to cDNA. The primers used in this experiment were listed in Table 1. The fluorescent quantitative PCR kit (TaKaRa) was used to perform RT-PCR and 2-ΔΔCT was used to calculate the relative gene expression. α-tublin was used as an internal reference.
Western blotting
The protein concentrations were determined using the bicinchoninic acid protein assay kit (Beyotime, China). All protein concentrations were adjusted to 2 mg/mL, and then separated by 8% SDS-PAGE. Then, the proteins were transferred to a nitrocellulose membrane (731809, Millipore, USA) and blocked with 5% skimmed milk for 1 h, and the membrane was sequentially incubated with a primary antibody and a horseradish peroxidase-conjugated secondary antibody. Finally, the enhanced chemiluminescence method was used to detect protein levels.
siRNA knockdown of SIRT1 expression
HaCaT cells in the logarithmic phase were incubated in six-well plates at a density of 5×104 cells/mL overnight. The next day, HaCaT cells were transfected with siRNAs (Table 2) using Lipofectamine 3000 (L3000015, Thermo Fisher Scientific), and incubated for 24 h in accordance with the manufacturer’s instructions. Then, fresh media was added, and the corresponding stimulant was added to the media for 48 h.
Statistical analysis
Data are presented as the mean ± S.D. of at least three independent experiments. One-way analysis of variance was performed using SPSS 19.0 software (SPSS Inc., Chicago, IL, USA) to determine the statistical significance of the results. P < 0.05 was considered statistically significant.