Interleukin 6 (il 6)

The tissue sections were deparaffinized and dehydrated by fractionation using xylene and ethanol. The sections were incubated for 1h at room temperature or overnight at 4°C with the following primary antibodies: IL-6 (anti-rabbit, D220828, Sangon, Shanghai, China), P-STAT3 (Tyrosine 705, #9145, CST), MMP2 (sc-8835, Santa Cruz), MMP9 (sc-6841, Santa Cruz), α-smooth muscle actin (α-SMA, Clone 1A4, DAKO) and CD68 (#76437, CST). After washing with PBS, tissue sections were incubated with biotinylated secondary antibody for 90 min at room temperature. Then an avidin-biotin peroxidase complex was added for another 30 minutes. Then diaminobenzidine was added as a substrate that reacts with immune cells to make stain and then the tissue sections counterstained with hematoxylin.

Complete Freund’s adjuvant (CFA) was obtained from Chondrex, Inc. (USA). D-luciferin was purchased from Abcam (UK). Rabbit anti-BMP3, anti-TNF-a, and anti-TIMP metallopeptidase inhibitor 1 (TIMP-1) antibodies were obtained from Abcam (UK). Rabbit anti-MMP-3 and anti-MMP-9 antibodies were acquired from Merck Millipore (USA). Rabbit anti-IL-6, anti-IL-1β, and anti-IL-17A were purchased from Bioworld (USA). The rat anti-β-actin antibody was purchased from CST (USA). A horseradish peroxidase (HRP)-labeled goat anti-rabbit immunoglobulin (IgG) was procured from Zhongshan Biotechnology Corporation (China). BMP3, MMP-3, MMP-9, TIMP-1, TNF-a, IL-6, IL-1β, IL-17A, CCL-2, CCL-3, VCAM-1, and β-actin primers were synthesized by the Shanghai Sangon Biological and Technological Company (China).

Resveratrol ≥99% and sodium tetrachloroaurate (III) dihydrate (NaAuCl4) ≥99% were purchased from Macklin Biochemical Co., Ltd. (Shanghai, China). GSH and MDA kits were purchased from Solarbio Bioscience & Technology Co., Ltd. (Beijing, China). Fetal bovine serum (FBS) and Dulbecco’s modified Eagle’s medium (DMEM) media were purchased from Thermofisher Scientific (Shanghai, China). Cell Counting Kit-8 (CCK-8) was from Bioground Bioscience & Technology Co., Ltd. (Chongqing, China). DCFH-DA was from Beyotime Biotechnology (Shanghai, China). EX-527 was obtained from Selleck (Shanghai, China). The primer for siSirt1 was designed by Sangon Biotech Co., Ltd. (Shanghai, China). Antibodies against p16, p21, β-actin, Sirt1, and Nrf2 were purchased from Abclonal Technology (Wuhan, China). BAX and BCL-2 were obtained from Abcam Plc (Shanghai, China). RT-qPCR amplification primers for GAPDH, IL-1β, IL-6, CXCL8, MMP1, TGFβ, Cryga, and Cryba1 were made by Sangon Biotech Co., Ltd. (Shanghai, China).

Male C57BL/6J mice were obtained from GemPharmatech (Nanjing, China) at ages ranging from 4 to 6 weeks old. Mice were housed in a pathogen-free facility, with free access to autoclaved water and were maintained on a 12-h light/dark cycle. Body weight was recorded weekly. All mice were fed normal chow diet unless otherwise indicated. For high-fat feeding, a 60% (by calories) fat diet (XTHF60, irradiated; Xietong Shengwu, Nanjing, China) was used. After 1 week of accommodation, mice were administrated with corresponding drugs. Metformin (1 mg/kilogram body weight per day, i.g.) was used as positive control. Phillyrin was gavaged at the concentrations of 25 and 50 mg/kilogram body weight daily. Control mice were gavaged with vehicle (0.1% w/v carboxymethylcellulose sodium). Phillyrin and Metformin were purchased from Sigma-Aldrich (Shanghai, China). Wildtype Male C57BL/6J mice fed on normal diet were used for the experiments of IL-6 (Sangon Biotech, Shanghai, China) injection into perigonadal white adipose tissue (gWAT). 1 μg of IL-6 dissolved in 50 μl PBS was injected into per depot of gWAT per mouse, after the mice were anesthetized with Zoletil 50 (50 mg/kg, Virbac, France) and the abdomen was surgically opened. All animal procedures were approved by the Institutional Animal Care and Use Committee (IACUC) at Anhui University of Chinese Medicine.