Modulation of Inflammatory Responses in Skin and Mast Cells

A human immortalized keratinocyte cell line (HaCaT cells) was provided by Korea Institute of Oriental Medicine (Daegu, Korea) and maintained in high glucose DMEM medium (Welgene Inc., Gyeongsangbuk, Korea) supplemented with 10% FBS and 1% antibiotics (10,000 μg/ml of streptomycin and 10,000 units/ml of penicillin) (Invitrogen Inc., Carlsbad, CA, USA) and incubated at 37°C with 5% CO₂. After serum-starvation for 24 h, HaCaT cells were pretreated with SMGGT (10 and 100 μg/ml), puerarin, paeoniflorin (1, 10 μM), or DEX (10 μM) for 1 h and then incubated with TNF-α and IFN-γ (TI; 10 ng/ml each) for the indicated times. Recombinant human TNF-α and IFN-γ were obtained from Koma Biotech Inc. (Seoul, Korea).
A human mast cell line (HMC-1 cells) was purchased from Merck Millipore (Darmstadt, Germany) and grown in Iscove's Modified Dulbecco's Medium (IMDM) (Merck Millipore) supplemented with 10% FBS, 1% antibiotics, and 1.2 mM 1-thioglycerol (Sigma-Aldrich, St. Louis, MO, USA), at 37°C with 5% CO₂. The cells were stimulated with 10 μM SP for 48 h, followed with 1 nM CRH for 24 h, as described previously [21 (link)]; SMGGT (10 and 100 μg/ml), puerarin, paeoniflorin (1, 10 μM), or DEX (10 μM) was treated 1 h before addition of CRH. SP and CRH were obtained from Sigma-Aldrich (St. Louis, MO, USA).