Nf κb

The lower lobe of the left lung was homogenized in lysis buffer, and the proteins were collected. For the detection of Nrf2 and NF-κB, the cytoplasmic component and nuclear component were isolated by treating a nuclear protein extraction kit (Beyotime Biotechnology, Shanghai, China) and centrifuged at 12,000 g for 10 min at 4°C. After the protein concentration was measured by a BCA kit, 50-μg protein samples were separated by SDS-PAGE and transferred onto a PVDF membrane. The membrane was blocked in 5% non-fat dry milk, followed by incubation with pro-caspase-3 (1:8,000, Abcam, United States), cleaved-caspase-3 (1:500, Abcam, United States), pro-caspase-9 (1:800, Abcam, United States), cleaved-caspase-9 (1:1,000, Abcam, United States), Nrf2 (1:5,000, Abcam, United States), and NF-κB (1:3,000, Abcam, United States) primary antibodies overnight at 4°C. After being washed and incubated with secondary antibody (1:5,000, Zhongshan Golden Bridge Biotechnology, Beijing, China) at room temperature for 1 h, the proteins were visualized with the enhanced chemiluminescence reagent (GE Healthcare Bio-Sciences, Pittsburgh, PA, United States), analyzed using ImageJ version 1.61 software (National Institutes of Health, Bethesda, MD, United States) and normalized to β-actin and Lamin B.

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.

Cell lysate was prepared using RIPA (Beyotime) or Pierce IP lysis buffer (Thermo fisher scientific) containing protease inhibitor cocktail. Protein quantification was performed by BCA assay (Beyotime). Membrane was blocked in 5% skin‐milk for 1 hour and then incubated with primary antibody overnight at 4C. Horseradish peroxidase‐conjugated secondary antibody (Beyotime) was then added for 1 hour at room temperature. Briefly, protein supernatant for Co‐IP was incubated with Protein A/G PLUS‐Agarose (Santa Cruz Biotechnology) beads for 3 hour at 4°C. The supernatant was transferred to a new tube, added anti‐ZHX2 or anti‐IgG antibodies, and incubated for 1 hour at 4°C. The mixture was incubated with beads under rotary agitation overnight at 4°C, then centrifuged and discarded the supernatant. Beads were collected and washed for three times. The 2 × loading‐buffer was added to beads and the mixture was heated at 100℃ for 10 minutes. Protein‐antibody complexes were determined by immunoblot. The following primary antibodies were used: ZHX2(GeneTex), NF‐κB (CST), Tubulin (Beyotime), and IgG (ABclonal).