THP-1 cells were purchased from the cell bank of the Chinese Academy of Sciences (Shanghai, China) and cultured in RPMI1640 medium (HyClone, Logan, UT, USA) containing 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) and 10×10⁴ U/ml penicillin/streptomycin (HyClone). Cells were cultured in an incubator at 37°C with 5% CO₂ and 95% O₂.

Transwell chambers used were ECM550 (Chemicon, Temecula, CA, USA). THP-1 cells with good growth conditions were made into single cell suspensions and 1×10⁵ (total volume of 200 µl) cells were added to the upper chamber. A volume of 300 µl of serum-containing culture medium with different concentrations of recombinant human HMGB1 (R&D Systems, Inc., Minneapolis, MN, USA) was added to the lower chamber and incubated for 24 h at 37°C. Then, the medium in the lower chamber was aspirated, sodium chloride-alcohol was added, and washed after fixing for 10 min. Then, 0.1% crystal violet (Sigma-Aldrich, St. Louis, MO, USA) was added to the lower chamber and stained for 10 min. Cells on the membrane of the upper chamber were carefully wiped, while cells on the basement membrane of the lower chamber were counted in 6 view fields randomly selected under a ×200 optical microscope. The average was recorded as the number of THP-1 cells passing through the artificial basement membrane. Different concentrations of GL (Sigma-Aldrich) were added to the culture fluid in the lower chamber as treatment before the 24 h culture.

Staurosporine (STS) (300 nM) with or without different concentrations of HMGB1 was added to the THP-1 cell culture medium and incubated for 24 h. After centrifugation and resuspension in PBS, 5–10×10⁴ cells were stained with 200 µl Annexin V-fluorescein isothiocyanate (FITC) and 10 µl propidium iodide (PI) following secondary centrifugation. Flow cytometry was performed by using a FACSCalibur system (Becton Dickinson, Franklin Lakes, NJ, USA) after incubation at room temperature in the dark for 10–20 min and ice bath. The STS and apoptosis detection kit Annexin V-FITC were obtained from Beyotime Institute of Biotechnology (Shanghai, China). The scatter plot was divided into four quadrants: The left lower quadrant was defined as viable cells (‘low’ FITC and ‘low’ PI signal), the right upper quadrant was defined as necrotic cells (‘high’ FITC and ‘high’ PI signal), and the right lower quadrant was defined as apoptotic cells (‘high’ FITC and ‘low’ PI signal). The percentage of Annexin V-positive cells in the total cells of the scatter plot was used to determine the results. Different concentrations of GL were added to the samples prior to the one day incubation for the intervention experiment.

Cellular proteins were extracted using lysis buffer, and the concentrations of protein were measured using a BCA Protein Assay kit (Thermo Fisher Scientific, Inc.). For the detection of NF-κB level in nucleus, nuclear protein was extracted using nuclear extraction kit (Epigentek, Farmingdale, NY, USA) in accordance with the manufacturer's instructions. Equal protein amounts were fractionated in 5–8% SDS-PAGE gels and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). After blocking with 5% skimmed milk powder, the membranes were incubated with the following primary antibodies at 1:1,000 dilution: Cleaved caspase-3, PARP, phosphorylation of IκBα (p-IκBα), IκBα, NF-κB p65, phosphorylation level of ERK1/2 (p-ERK1/2), ERK1/2, and Mcl-1 (all from Cell Signaling Technology, Beverly, MA, USA), and HMGB1, β-actin and Lamin B1 (all from Santa Cruz Biotechnology, Inc., Dallas, TX, USA) overnight at 4°C. The membranes were then incubated with HRP-conjugated rabbit or mouse secondary antibodies (Cell Signaling Technology) at room temperature for 2 h after washing. Immunoblots were detected by using an enhanced chemiluminescence reagent (Tiangen Biotech, Co., Ltd., Beijing, China) and imager (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Gray value was analyzed with the software ImageJ, and the gray coefficient ratio was calculated.

Total RNA was isolated from THP-1 cells using the TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.). Reverse transcription was performed using a First Strand cDNA Synthesis kit (Fermentas, ON, Canada). PCR amplification was performed using a One Step SYBR-Green I Quant qRT-PCR kit (Tiangen Biotech, Co., Ltd.) on an ABI 7000 real-time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.). Table I shows the primers used for MCP-1 and β-actin synthesized by Invitrogen (Thermo Fisher Scientific, Inc.) for PCR. PCR parameters were 5 min at 94°C, followed by 40 cycles of 30 sec at 94°C, 30 sec annealing at 60°C for MCP-1 or 55°C for β-actin, and 30 sec at 72°C, ending at 72°C for 10 min. The 2^−∆∆Cq method (10) was used to calculate the relative levels of MCP-1 mRNA, and β-actin expression was used for normalization.

Data are expressed as the mean ± SEM. Differences between groups were evaluated using one-way ANOVA followed by the Newman-Keuls test, using SPSS version 16.0 software (SPSS, Inc., Chicago, IL, USA). Statistical significance was established when P<0.05.

THP-1 cells were treated with different concentrations of HMGB1 (0.1, 0.5, and 1 µg/ml). The results of Transwell assays showed that HMGB1 significantly increased the number of migrated cells, especially at higher concentrations (Fig. 1A). GL at all concentrations tested (10, 50, and 100 µg/ml) suppressed the HMGB1-induced monocyte migration, particularly at 50 and 100 µg/ml (Fig. 1B).

THP-1 cells were treated with STS and HMGB1 (0.1, 0.5, and 1 µg/ml). Fig. 2A shows that the control group had a lower apoptosis rate, and STS significantly induced apoptosis. HMGB1 at 0.5 and 1 µg/ml suppressed STS-induced apoptosis, and GL at 50 and 100 µg/ml significantly improved the inhibition of apoptosis by HMGB1 (Fig. 2B). Furthermore, we explored effects of HMGB1 and GL on key factors in apoptotic pathway. As shown in Fig. 2C and D, STS significantly increase the level of cleaved caspase-3 and PARP. HMGB1 suppressed the STS-induced cleavage of caspase-3 and PARP, and GL significantly increased the cleavage at 50 and 100 µg/ml.

HMGB1 promotes monocyte recruitment by activating the RAGE/NF-κB signaling pathway (3). p-IκBα results in the activation of nuclear translocation of NF-κB. The levels of p-IκBα and nuclear NF-κB p65 were detected to evaluate the effect of GL treatment on the activation of NF-κB. Fig. 3 shows that HMGB1 increased the level of p-IκBα and nuclear NF-κB p65, whereas GL decreased this level by suppressing the function of HMGB1. MCP-1 has the ability to recruit and activate particular leukocytes and is a potential downstream effector of NF-κB (11). RT-qPCR was performed to detect the effect of HMGB1, the NF-κB inhibitor QNZ (100 nM; Selleck Chemicals, Houston, TX, USA), and GL on the expression of MCP-1. HMGB1 significantly increased the mRNA expression of MCP-1, whereas QNZ and GL remarkably suppressed the induction of MCP-1 expression by HMGB1 (Fig. 4).

Since HMGB1 inhibits apoptosis of monocytes by activating the TLR4/MAPK/ERK signaling pathway (3), we further investigated the effect of GL on this pathway. HMGB1 significantly increased the p-ERK1/2, whereas GL suppressed the HMGB1-induced activation of ERK1/2 (Fig. 5). It has been reported that the protein degradation of Mcl-1 is inhibited by ERK (12). Therefore, activation of the ERK pathway could prevent the protein degradation of Mcl-1. We speculated that HMGB1 may inhibit the degradation of Mcl-1 via the MAPK/ERK pathway, and further antagonize apoptosis. Fig. 6 shows that HMGB1 significantly increased the level of Mcl-1 compared to that in the STS treated group. The level of Mcl-1 increased by HMGB1 was remarkably decreased by the ERK1/2 inhibitor SCH772984 (SCH) (10 nM; Medchemexpress LLC, Princeton, NJ, USA) and GL.