Helenalin

Forty-eight hours after seeding, RSFs were washed with PBS 2 times and brought to serum-free conditions overnight prior to beginning the experiment. To investigate MMP-1 and MMP-3 mRNA expression and protein secretion, RSFs were treated with various concentrations of HAoligos (0–500 μg/ml) in serum-free DMEM for 12 or 24 hours. In some experiments, cells were pretreated with 5 μg/ml anti-CD44 or 5 μg/ml anti-TLR-4 antibody for one hour before HAoligo treatment. To investigate intracellular signaling pathways, RSFs were pretreated with anti-TLR-4 antibody followed by 250 μg/ml HAoligos for 0–120 min. For signal inhibition assays, RSFs were pretreated with inhibitors for NF-κB (Helenalin; Merck KGaA, Darmstadt, Germany) or p38 MAPK (SB203580; Merck KGaA) for one hour, followed by the addition of 250 μg/ml HAoligos for 24 hours in the presence or absence of each inhibitor. To examine the effects of HMW-HA on HAoligo-induced MMP mRNA expression, RSFs were pretreated with 250 μg/ml HAoligos for 12 hours and followed with 250 μg/ml HAoligos or 1 mg/ml HMW-HA (600–1,200 kDa; Seikagaku Co., Tokyo, Japan) for 12 hours.

Human peripheral blood mononuclear cells (PBMC) were isolated from healthy donors by Ficoll-Paque PLUS (GE Healthcare, Uppsala, Sweden) according to manufacturer's suggestions. CD14⁺ cells were further purified from PBMC by anti-CD14 microbeads and magnetically activated cell sorting (MACS) system (Miltenyi Biotech, Auburn, CA) according to manufacturer's instruction. PBMC and purified CD14⁺ cells (purity >90%) were cultured in RPMI 1640 medium (Sigma-Aldrich, USA) supplemented with 10% heat-inactivated Fetal bovine serum (FBS) (Sigma-Aldrich, USA) and penicillin/streptomycin (100 units/ml) (Invitrogen, CA, USA). For isolation of neutrophils from healthy donors, leukocytes (including neutrophils and PBMC) were separated from red blood cells (RBC) by differential sedimentation using 1.5% dextran in PBS. Neutrophils were separated from PBMC by Ficoll-Paque PLUS gradient method and further separated from the remaining RBC in the pellet by hypotonic lysis method (purity>90%) [57] (link). THP-1 cells (ATCC number: TIB-202) was maintained in RPMI 1640 supplemented with 10% FBS and penicillin/streptomycin (100 units/ml). For experiments of cell signaling pathway, inhibitors including PD98059, SP600125, SB203580, LY294002, Helenalin and MG132 were purchased from Calbiochem (Merck Millipore, Germany) and dissolved in dimethyl sulfoxide (DMSO) (Sigma-Aldrich, USA) for cell experiments.

Matrine was obtained from Sigma-Aldrich (St. Louis, MO, USA) and was dissolved in dimethyl sulfoxide (DMSO) for cell culture. Fetal bovine serum (FBS), penicillin and streptomycin were all purchased from Gibco Life Technologies (Carlsbad, CA, USA). Helenalin was purchased from Sigma-Aldrich. Mouse monoclonal anti-nuclear factor-κ (NF-κB) p50 (sc-271908), mouse monoclonal anti-NF-κ p65 (sc-71676), mouse monoclonal anti-β-actin (sc-376421) and rabbit polyclonal anti-histone H1 (sc-67324) were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Rabbit polyclonal anti-MMP-2 (#4002) and rabbit polyclonal anti-MMP-9 (#2270) antibodies were purchased from Cell Signaling Technologies (Danvers, MA, USA).