ZGC extract preparation
ZGC was purchased from China Resources Sanjiu Medical & Pharmaceutical Co., Ltd, the catalog number was 1912002S. The mixture was resolved in water by sonication . The extracts were dried by lyophilization and stored at - 80 °C at the final concentration at 100mg/mL.
ATDC5 cell was purchased and shipped from American Type Culture Collection (ATCC, Manassas, VA) and growth in DMEM/F12 medium containing 100 IU/mL penicillin, 100 μg/mL streptomycin and 10% fetal bovine serum. ATDC5 were incubated at 37 °C incubator with 5% CO2. Once the cells were 70% confluent, the medium was supplemented with 1% ITS and to induce chondrocyte-like cells. Chondrocyte-like cells were pre-treatment with Lipopolysaccharide (LPS, 1μg/mL) for 24 hours before drug or herbal extract presence. LPS was obtained from Sigma (St. Louis, MO).
PI-Annix staining assay
Cells were seeded in 35-mm culture plates and allowed to grow for 24 hours in medium before drug treatment. The Annexin V-FITC/PI Detection kit was employed using flow cytometry (BD Biosciences, Franklin Lakes, NJ). Briefly, cells were washed by PBS twice and then harvested. Cells were incubated in 100 μL of binding buffer, containing Annexin-V/FITC and propidium iodide (PI), for 15 min at room temperature and kept in dark. The samples were automatically acquired using the loader with acquisition criteria of 10,000 events for each tube, and the quadrants were set according to the population of viable. The results were analyzed using the FlowJo v10.6 software.
Real time PCR
The transcriptional levels of IL-1β, IL-6 and TNF-α were detected in LPS-stimulated chondrocyte-like cells. After the ZGC treatment for 48 hours, total RNA was isolated by RNAzol reagent (Molecular Research Center, Cincinnati, OH) and then reversed transcribed into cDNAs by using M-MLV (moloneymurine leukemia virus) reverse transcriptase according to the manufacturer’s suggestions (Sigma). Real-time PCR was employed here by using FastStart Universal SYBRGreen Master (ROX) according to the manufacturer’s instructions (Roche Applied Science, Mannheim, Germany). The sequences of primers were shown here: 5′-AAA TAC CTG TGG CCT TG-3′ (sense primer, S) and 5′-TTA GGA AGA CAC GGA TTC-3′ (antisense primer, AS) for murine IL-1β (NM_008361); 5′-GGA GTA CCA TAG CTACCT GG-3′ (S) and 5′-CTA GGT TTG CCG AGT AGA TC-3′ (AS) for murine IL-6 (NM_031168); 5′- AGT GAC AAG CCT GTA GCC -3′ (S) and 5′-AGG TTG ACT TTC TCC TGG-3′ (AS) for murine TNF-α (NM_013693); 5’-GAT GAG GCT GCG GAA GAA GG-3’ (S) and 5’-GCG AAG GCG TGG GTT CAG-3’ (NM_022415). Glyceraldehyde 3-phosphate de-hydrogenase (GAPDH) was used as an internal control, and the sequences were shown here: 5′-AAC GGA TTT GGC CGT ATT GG-3′ (S) and 5′- CTT CCC GTT CAG CTC TGG G-3′(AS) (NR_0215885). SYBR green signal was revealed by ABI 7500 Fast Real-Time PCR system (Applied Biosystems, Foster City, CA). Transcript levels were quantified by using ΔCt value method [14, 20], where the values of target genes were normalized by GAPDH in the same sample. Dexamethasone (Dex, 10 μM) was obtained from Sigma working as positive control in all experiments.
Protein expression of cytokines
Protein expressions of proinflammatory cytokines after drug treatment for 2 days were revealed by Milliplex® technology (Millipore, MA). Supernatants of culture medium were collected to measure the concentrations of cytokines (IL-1β, IL-6 and TNFα) using Milliplex® technology, followed the manufacturer’s instruction.
Fluorometric measurements of reactive oxygen species (ROS) were utilized CellROX Deep Red Fluorescence Flow Cytomer detection kit (BD Biosciences) under manufacturer’s suggested protocol. Briefly, cells at were incubated in 100 μL of binding buffer for 15 min at room temperature. Samples were automatically acquired using the loader with acquisition criteria of 10,000 events for each tube, and the quadrants were set according to population of viable. The cytometer results were analyzed with Flowjo v7.6 software.
Cell lysate were subjected to SDS-PAGE in revealing protein expressional levels of target genes. After transferring the target proteins to membranes, the membranes were incubated with anti-iNOS (CST, Danvers, MA) at 1:5,000 dilutions, anti-COX-2 (CST) at 1 : 5,000 dilutions, anti-PGES2 (Santa Cruz, Dallas, TX) at 1:5000 dilutions and anti-GAPDH (Santa Cruz) at 1 : 5,000,000 dilutions at 4 °C for overnight. Following incubation in horseradish peroxidase (HRP)-conjugated secondary antibodies for 3 hours at room temperature, the immune-complexes were visualized by the enhanced chemiluminesence (ECL) method (Amersham Biosciences, Piscataway, NJ). The band intensities were calculated and analyzed by Image J.
Laser confocal assay
The translocation of NF-κB p65 subunit was activated by treatment of 1 µg/mL of LPS for 24 hours and then presence with ZGC for another 48 hours. Then, cells were fixed for 10 min in 4% methanol-free paraformaldehyde. Antibodies were diluted in PBS containing 2.5 % fetal bovine serum, and 0.1% Triton X-100 (Sigma). NF-κB p65 subunit antibody was diluted 1:500 in PBS. Samples were incubated for overnight at cold room. Then, 1:1,000 dilution of secondary FITC-conjugated F(ab’)2 fragment donkey anti-rabbit IgG antibody (Jackson Laboratories, West Grove, PA) was added, incubated at room temp for 3 hours, in dark. After incubating, the cells were washed for 3 times by PBS. Before performing assay, 1: 5,000 dilution of DAPI was added to stain nucleus. Fluorimetric measurements were performed using an Olympus Fluoview FV1000 laser scanning confocal system (Olympus America, Melville, NY) mounted on an inverted Olympus microscope, equipped with a 63X objective.
Statistical analysis and other assays
Protein concentrations were measured by Bradford’s method (Herculues, CA). Statistical tests have been done by using one-way analysis of variance. Data were expressed as Mean ± SEM, where n = 3-4. Statistically significant changes were classified as significant (*) where p < 0.05, more significant (**) where p < 0.01 and highly significant (***) where p < 0.001 as compared with control group. Significant (^) where p < 0.05, more significant (^^) where p < 0.01 and highly significant (^^^) where p < 0.001 are compared with positive control group.