Sirt1

Sweroside (purity > 98%, Figure 2A) was obtained from National Institutes for Food and Drug Control (Beijing, China). Fetal bovine serum (FBS) and phosphate buffer saline (PBS) were acquired from Servicebio Company (Wuhan, China). DMEM, DMSO, trypsin, the RIPA Lysis buffer and the PVDF membrane were obtained from Solarbio Company (Beijing, China). The annexin V-FITC apoptosis detection kit was obtained from Vazyme Biotech Company (Nanjing, China). Hoechst 33342, NAM, SIRT1, NF-κB, β-actin and the proliferating cell nuclear antigen (PCNA) antibodies were from Beyotime Institute of Biotechnology (Shanghai, China). The TNF-α, IL-1β, IL-6, IL-10, MnSOD, FOXO1, COX-2 and i-NOS antibodies were obtained from Proteintech Company (Wuhan, China). The ROS assay kit, CCK-8 assay kit, NO assay kit, cell cycle and apoptosis analysis kits were from Beyotime Institute of Biotechnology (Shanghai, China). The PGE2 Elisa Kit was purchased from Hui Jia Company (Xiamen, China). The other chemicals were of analytical grade and were commercially available.

2′-O-GH (purity ≥ 98%) was isolated from Pyrola [P. incarnata Fisch.], and the chemical structure was identified in our laboratory (Yao et al., 2013 (link), 2015 (link)). A 10-mM stock solution of 2′-O-GH was prepared in DMSO and stored at -80°C.
Polyclonal antibodies against JNK, phospho-JNK (Thr183/Tyr185), p38 MAPK, phospho-p38 MAPK (Thr180/Tyr182), p42/p44 MAPK, phospho-p42/44 MAPK (Thr202/Tyr204), phosphor-IKKα/β (Ser176/177), NF-κB p65, SIRT1, β-actin and inhibitor compounds U0126 (a ERK inhibitor), SB203580 (p38 MAPK inhibitor), and SP600125 (JNK inhibitor) were purchased from Beyotime Institute of Biotechnology (Beijing, China). DMSO, LPS (from Escherichia coli O111: B4), NAC and MTT were obtained from Sigma (St. Louis, MO, United States). DEX was supplied by Xi’an Lijun Pharmaceutical Company Limited (Xi’an, China). Other reagents and chemicals were purchased from Beijing Chemical Reagents Co. (Beijing, China). Secondary antibodies were obtained from Beyotime Institute of Biotechnology (Beijing, China). Deionized water was purified by a Milli Q Water Purification system from Millipore (Millipore Corp., Bedford, MA). PVDF membrane was purchased from Millipore (Millipore Corp., Bedford, MA).

Radioimmunoprecipitation assay buffer with protease and phosphatase inhibitors was used to lyse the NR8383 cells. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (10%) was used to separate the proteins from 20 μg of cell lysate, and then the proteins were transferred to polyvinylidene fluoride membranes. Primary antibodies against NLRP3, ASC, P10, SIRT1 and β-actin (Beyotime, Wuhan, China) were used to blot the membranes, and then the corresponding secondary antibodies were applied (horseradish peroxidase-labeled antibody; Beyotime). Pierce^TM ECL Plus Western blot substrate was used to visualize the immunoblots.