HaCaT cells (Shanghai Blowing Applied Biotechnology Co., Ltd., Shanghai, China; STR profile confirmed as appropriate) were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, and 1% penicillin and streptomycin (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany), and were incubated at 37°C in a humidified incubator containing 5% CO₂. Recombinant human TNF-α was purchased from BioLegend, Inc. (San Diego, CA, USA). APS (75.6%) was supplied by the Chinese Academy of Sciences, Shanghai Institute of Materia Medica (Shanghai, China).

The viability of HaCaT cells was investigated using Cell Counting Kit-8 (CCK-8; Dojindo Molecular Technologies, Inc., Kumamoto, Japan). Cells were seeded into a 96-well plate at a density of 5.0×10³ cells/well and were incubated for 24 h at 37°C in 5% CO₂. Subsequently, cells were treated with TNF-α (10 ng/ml), various concentrations of APS (20–80 µg/ml) or a combination of TNF-α (10 ng/ml) and APS (20, 40 and 80 µg/ml) for 24 h at 37°C in 5% CO₂. CCK-8 solution (10 µl) was then added to each well, and the plates were incubated for an additional 2 h at 37°C. The control group was treated with an equal amount of PBS. Absorbance values at 450 nm were determined for each well using a microplate reader. Cell proliferation rate=(OD_{24 h}-OD_{0 h})/OD_{0 h} ×100%. All assays were carried out in triplicate.

HaCaT cells were treated with either TNF-α or a combination of TNF-α and various concentrations of APS, and supernatants were collected 24 h post-treatment. Concentrations of interleukin (IL)-8 (cat. no. D8000C) and IL-12 (cat. no. M1270) were determined using ELISA kits (RayBiotech, Inc., Norcross, GA, USA). Briefly, 100 µl of each standard and supernatant sample were added to a 96-well plate coated with anti-Human IL-8 or IL-12 and incubated overnight at 4°C with gentle agitation. Plates were subsequently washed four times with wash buffer. Wells were then incubated for 1 h with IL-8 or IL-12 specific biotinylated antibodies at room temperature, and were rinsed four times with wash buffer. Following this, cells were treated with diluted streptavidin solution (100 µl/well) for 45 min at room temperature, washed four times with wash buffer, and further incubated for 30 min at room temperature with 100 µl TMB One-Step substrate reagent in the dark. The plates were quenched with stop solution and absorbance values were detected at 450 nm using a PowerWave XS spectrophotometer (BioTek Instruments, Inc., Winooski, VT, USA). ELISA analyses for each sample were repeated in triplicate. All assays were carried out in triplicate.

Protein lysates were prepared using radioimmunoprecipitation assay lysis buffer (Beyotime Institute of Biotechnology, Haimen, China) with a protease/phosphatase inhibitor cocktail (Cell Signaling Technology, Inc., Danvers, MA, USA). Antibodies for IL-8 (cat. no. 94853S; 1:1,000), NF-κB inhibitor-α (IκBα; cat. no. 4814S; 1:1,000), NF-κB (p65; cat. no. 8242S; 1:1,000) purchased from Cell Signaling Technology, Inc, and IL-12 (cat. no. ab9992; 1:1,000) purchased from Abcam, (Cambridge, MA, USA). GAPDH mouse monoclonal antibody (cat. no. D190636; 1:1,000) was purchased from Sangon BioTech Co., Ltd. (Shanghai, China). The protein concentrations were determined through BCA Protein Assay kit (Vazyme, Piscataway, NJ, USA). Samples with equal amounts of protein (25 µg) were fractionated on 10% SDS-PAGE and were then transferred to polyvinylidene fluoride membranes. Following blocking with 5% non-fat milk for 1.5 h at 25°C, the membranes were incubated with primary antibodies for at 4°C overnight. Membranes were then washed with PBS and incubated with goat anti Rabbit IgG horseradish peroxidase (cat. no. ab6721; 1:1,000) for another 2 h at 25°C. Blots were developed with SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific, Inc., Waltham, MA, USA) and the images were obtained by ImageQuant LAS 4000 (GE Healthcare Life Sciences, Little Chalfont, UK). All assays were carried out in triplicate.

Extraction of RNA from cell lysates was performed using an RNeasy kit (Qiagen GmbH, Hilden, Germany). RNA was then subjected to cDNA synthesis using PrimeScript RT reagent kit (Takara Bio, Inc., Otsu, Japan). Synthesized cDNA was analyzed by RT-qPCR using SYBR Premix Ex Taq (Takara Bio, Inc.) according to manufacturer's protocol, the PCR conditions consisted of 95°C for 30 sec, followed by 40 cycles of amplification (95°C for 3 sec and 60°C for 30 sec). Bio-Rad CFX96 Touch™ Real-Time PCR Detection system (Bio-Rad Laboratories, Inc., Hercules, CA, USA) was performed. The fold-change was determined as 2^−∆∆Cq using β-actin as an internal control (21). The primer sequences are as follows: NF-κB (p65), forward 5′-ATCAATGGCTACACAGGA-3′, reverse 5′-CCCGTGAAATACACCTCA-3′; and β-actin forward 5′-CATGTACGTTGCTATCCAGGC-3′ and reverse 5′-CTCCTTAATGTCACGCACGAT-3′. All assays were carried out in triplicate.

All data were analyzed using Prism 5.0 software (GraphPad Software Inc., La Jolla, CA, USA) and expressed as the means ± standard error of the mean. Significant differences between groups were statistically analyzed using one-way analysis of variance followed by the Newman-Keuls method. P<0.05 was considered to indicate a statistically significant difference. All assays were carried out in triplicate in the present study.

The viability of HaCaT cells was investigated to determine the effects of APS on HaCaT keratinocytes. The results demonstrated that compared with in the negative control (NC) group, cell viability was not altered following treatment with APS (20, 40 and 80 µg/ml) (Fig. 1).

Psoriasis is characterized by excessive proliferation of keratinocytes (14,15); therefore, the majority of treatment strategies to alleviate psoriasis are via suppression of the proliferation of keratinocytes. To investigate the effects of APS on psoriasis, HaCaT cells were treated with various concentrations of APS (20, 40 and 80 µg/ml) for 24 h. Furthermore, TNF-α is involved in the overproliferation of keratinocytes (22). The results of the present study demonstrated that HaCaT cells treated with 10 ng/ml TNF-α exhibited a significantly increased rate of cell proliferation compared with in the NC group (Fig. 2). In addition, the results revealed that TNF-α-stimulated cells treated with APS exhibited significantly decreased rates of cell proliferation in a dose-dependent manner compared with cells treated with TNF-α alone (Fig. 2). These results suggested that APS inhibited the inflammatory factor-induced proliferation of HaCaT keratinocytes.

Inhibition of TNF-α regulates numerous inflammatory factors; therefore, TNF-α inhibitors have been approved for the treatment of patients with psoriasis (23). Following treatment with 10 ng/ml TNF-α, or a combination of 10 ng/ml TNF-α and increasing concentrations of APS (20, 40 and 80 µg/ml), for 24 h, IL-8 and IL-12 protein expression levels were detected in HaCaT cell lysates. The results of western blotting revealed that TNF-α treatment significantly enhanced the protein expression levels of IL-8 and IL-12 compared with in the NC group (Fig. 3A-C). Furthermore, cells additionally treated with APS exhibited significantly decreased levels of IL-8 and IL-12 in a dose-dependent manner compared with in cells treated with TNF-α alone (Fig. 3A-C).

Following treatment with TNF-α, cells exhibited enhanced levels of secreted IL-8 and IL-12 into the culture medium compared with in the NC group (Fig. 4A and B). In addition, in TNF-α-stimulated cells treated with increasing concentrations of APS, the secretion of IL-8 and IL-12 into the culture medium was significantly decreased in a dose-dependent manner compared with in cells treated with TNF-α alone (Fig. 4A and B).

NF-κB functions as a linker for dysregulated crosstalk between keratinocytes and immune cells in the pathogenesis of psoriasis (24). In patients with psoriasis, NF-κB signaling is normally activated by TNF-α; therefore, TNF-α inhibitory drugs such as etanercept may decrease p-p65 levels and subsequently attenuate symptoms of the disease (25). Following treatment with TNF-α, HaCaT cells exhibited increased expression levels of p65 mRNA compared with in the NC group; however; administration of APS attenuated this effect in a dose-dependent manner (Fig. 5).

Notably, following treatment with TNF-α, the protein expression levels of p-p65 were significantly increased in HaCaT cells compared with in the NC group; however, this effect was significantly attenuated following treatment with APS in a dose-dependent manner (Fig. 6A and B). In addition, HaCaT cells treated with TNF-α exhibited significantly decreased levels of IκBα, an inhibitory protein of NF-κB; however, this effect was significantly attenuated following treatment with APS in a dose-dependent manner (Fig. 6A and C).