The Inhibitory Mechanism Of Baicalein On Tumour Necrosis Factor-alpha-Induced Proliferation Of Human Rheumatoid Arthritis Fibroblast-like Synoviocytes

ObjectiveBaicalein, isolated from the root of the traditional Chinese herbal medicine Huangqin (Scutellaria baicalensis Georgi), has been shown to inhibit the proliferation andpromote the apoptosis of various cancer cells.it can also inhibits the production of TNF-a, IL-6, and IL-1β in human RAFLS, together with ketorolac tromethamine. Considering its anti-inflammatory properties, we examined its effects on tumor necrosis factor-alpha (TNF-a)-induced proliferation of human rheumatoid arthritis fibroblast-like synoviocytes (RAFLS) and its potential mechanisms.Materials and methods(1)Patients:FLSs were obtained from human synovial tissue of RA patientsundergoing total knee replacement surgery, who met the revised criteria of the American College of Rheumatology. Informed consent was obtained from all patients, and the experimental protocol was approved by the Department of Orthopedics, Jinling Hospital, School of Medicine, Nanjing University.(2) cell culture of RAFLS:Briefly, the fresh synovial tissues were removed of adipose tissues followed by vigorous washes in phosphate-buffered saline (PBS) containing penicillin and streptomycin to get rid of debris and blood cells. Then the synovial tissues were cut into small pieces in centrifuge tubes and digested with collagenase II for 5-6h. The cell suspension was filtered through a cell strainer (200 mesh), and the filtrate was centrifuged at 1200 rpm for about 10 min. The supernatant was discarded. The cell pellets were resuspended with Dulbecco’s modified Eagle’s medium containing 10% fetal calf serum at 37 C in a 5% CO2 humidified incubator.(3)CCK-8 cell proliferation assay:Briefly, cells were seeded in 96-well plates at 5x104 per well at 37 ℃ in a 5% CO2 humidified incubator for 24 h. Then the cells were stimulated with human recombinant TNF-a (0 or 0.1 ng/ml), baicalein (0,10,20, or 40 μM), and/or human recombinant MIF (0,12.5,25,50 ng/ml)for 24h, respectively. The effects of baicalein on TNF-a or MIF-induced proliferation of RAFLS were evaluated by CCK-8 assay according to the manufacturer’s instructions. Optical density (OD) was detected at 450nm with an enzyme-labeled instrument. Relative cell viability was calculated with the following formula:relative cell viability (%)= (OD (treatment group)-OD (blank group))/(OD (control group)-OD(blank group))× 100%.(4)Preparation of nuclear extracts:Nuclear extracts were prepared using cytoplasm-nucleoprotein extraction kit. Cells were processed with indicated treatments according to the manufacturer’s protocol followed by mixing with cytoplasm protein cracking fluid and centrifuged at 14,000 rpm for 5 min. The supernatant (cytoplasm protein) was discarded. The precipitation fraction was resuspended with nuclear cracking liquid and incubated for 30 min at 4 ℃ with occasionally vigorous shaking. The mixture was centrifuged at 14,000 rpm for 20 min to collect the supernatant (nuclear extracts) and stored at-80 ℃.(5)Western blot analysis.-RAFLS (5×105) were incubated with TNF-a (10 ng/ml) for 30 min in the presence or absence of baicalein (20 μM). After treatment, the cells were homogenized in the lysis buffer and centrifuged at 14,000 rpm for 15 min. The protein concentration in the supernatant was determined using Bio-Rad Protein Assay Kit. Protein samples adequately mixed in 5×loading buffer were boiled for 5 min and separated on 12% SDS-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane. Then the membranes were blocked with 5% non-fat milk in Tris-buffered saline with Tween-20 (TBST). The primary antibodies for phosphorylated extracellular regulating kinase (ERK) 1/2 (p-ERK1/2), total ERK 1/2, phosphorylated p38 (p-p38), total p38, phosphorylated c-Jun N-terminal kinase (p-JNK), total JNK, nuclear factor kappa B (NF-κB), subunit p65, and histonewere added and incubated overnight. The blots were washed for three times with TBST and incubated with horseradish peroxidase conjugated secondary antibody at room temperature for 1 h. After another 3 times washes with TBST, immunoreactive proteins were visualized by an enhanced chemiluminescent detection system. Blots were scanned with Fluor-S MAX Multilmager, and signal intensities were determined by Quantity One image software.(6)Electrophoretic mobility shift assay:RAFLs (5×105) were stimulated with TNF-a (5 ng/ml)in the presence or absence of baicaleinfor 1h. With prepared nuclear extracts, electrophoretic mobility shift assays (EMSA) were performed using the LightShift chemiluminescent electrophoretic mobility shift assay kit to detect the activation of NF-κB and AP-1. The synthetic oligonucleotides containing NF-κB binding site (5’-AGT TGA GGG GAC TTT CCC AGG C-3’) or AP-1 binding site (5’-CGC TTG ATG AGT CAG CCG GAA-3’) were used as probes, which were labeled with (y32P)-dATP afterwards. The DNA-protein binding reaction was conducted in a final volume of 20 ml containing binding buffer,10 mg of nuclear extracts and the biotin-labeled oligonucleotide for 30 min. The reaction mixtures were analyzed by electrophoresis on 4% poly acry lamide gels. Then the gels were dried and visualized by autoradiography.Result(1)Baicalein inhibits TNF-a-induced proliferation of RAFLS. (2)Baicalein inhibits MIF-induced proliferation of RAFLS. (3)Baicalein decreases TNF-a-mediated phosphorylation of ERK and p38 in RAFLS. (4)Baicalein attenuates TNF-a-induced NF-κB DNA binding activity.ConclusionOur data revealed that baicalein inhibited TNF-a-induced proliferation of RAFLS probably through inhibiting NF-κB activation pathway, decreasing the phosphorylation of MAPK/ERK/p38, and suppressing MIF signaling pathway.