LBPs (purity >95%, data not shown) were purchased from Ningxia Qiyuan Pharmaceutical Company (Yinchuan, China). Antibodies against GAPDH, lamin B and NF-κB were from Abcam (Cambridge, MA, USA). The anti-HSP60 antibody was from Stressgen (San Diego, CA, USA) and the anti-caspase 3 antibody was from Cell Signaling Technology, Inc. (Beverly, MA, USA). The proteinase inhibitor cocktails were from Merck Chemicals (Whitehouse Station, NJ, USA). HSP60 and TNF-α enzyme-linked immunosorbent assay (ELISA) kits were from Yaji (Shanghai, China) and eBioscience (San Diego, CA, USA), respectively. BCA and ECL kits were from Pierce (Rockford, IL, USA). Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum (FBS) were from Gibco (Grand Island, NY, USA). All the additional chemicals were purchased from Sigma (St. Louis, MO, USA), unless otherwise stated.

BV2 mouse microglial cells were cultured in DMEM supplemented with 10% FBS, penicillin (100 U/ml) and streptomycin (100 g/ml). Cultures were maintained at 37°C in a humidified incubator gassed with 95% O₂ and 5% CO₂. LBPs were dissolved in phosphate-buffered saline (PBS). The final concentration of PBS in the medium was <0.05% (vol/vol) and this demonstrated no effect on cell growth (data not shown). The cells were treated with the indicated concentrations of LBPs for 24 h following the administration of LPS (200 ng/ml).

Cells (3×10⁴ cells in 200 μl/well) were seeded in 96-well plates. An MTT assay was used to assess the percentage of viable cells following pharmacological treatments, as previously described (19). Briefly, the culture medium in the 96-well plates was replaced by 100 μl of fresh MTT solution (0.5 mg/ml) following the treatments. After 3 h of incubation at 37°C, the supernatant was removed and formazan salt crystals dissolved in 150 μl dimethyl sulfoxide (DMSO) were added to the cells. Cell viability was measured by the absorbance at 570 nm. The cell survival ratio was calculated by normalization to the control.

The levels of HSP60 and TNF-α in the culture medium were quantified according to the manufacturer's instructions. Absorbance was determined at 450 nm using a microplate reader.

The cells were washed with PBS three times and lysed with RIPA buffer. The protein concentration was determined using a BCA kit, according to the manufacturer's instructions. Equal quantities of protein were loaded and run on SDS/polyacrylamide gels and then transferred to PVDF membranes. The membranes were blocked with 5% dried milk and incubated with the primary antibodies in TBST overnight at 4°C. After being rinsed in milk-TBST, blots were incubated in the horseradish peroxidase-conjugated secondary antibodies. The target proteins were detected using the enhanced chemiluminescence (ECL) detection system and X-ray films.

Statistical differences were determined using a one-way ANOVA test. P<0.05 was considered to indicate a statistically significant difference. Data in the text and figures are presented as the mean ± standard error of the mean (SEM). n represents the number of experiments.

An MTT assay was performed to determine whether LBPs had an effect on the apoptosis of LPS-stimulated BV2 cells. The results demonstrated that the treatment of microglia with 1,000 μg/ml LBPs for up to 24 h did not increase the viability of LPS-stimulated BV2 cells (data not shown). However, lower concentrations of LBPs (200–800 μg/ml) significantly increased cell viability compared with the LPS group (Fig. 1). These results indicate that LBPs have a positive effect on the viability of LPS-stimulated BV2 microglia.

NF-κB regulates the expression of numerous proinflammatory factors (20). Therefore, we examined the NF-κB level following LBPs treatment. Fig. 2A shows that levels of the p65 subunit of NF-κB increased with LPS treatment, but was markedly inhibited by LBPs. Caspase 3 is the upstream protein of the NF-κB signaling pathway. Inhibition of caspase 3 prevents neuronal loss in brain diseases, including activated microglia. Thus, we examined the effects of LBPs on caspase 3 expression (Fig. 2B). Our results showed that caspase 3 expression was suppressed following LBPs treatment. NF-κB has been shown to initiate the transcription of the HSP60 stress protein gene, which elicits a potent proinflammatory response in innate immune cells. Results from a previous study have demonstrated that LPS increases HSP60 expression in BV2 microglial cells (6). The results of the present study also showed that HSP60 expression markedly increased following LPS treatment. However, LBPs treatment significantly inhibited this HSP60 enhancement (Fig. 2C). These results suggest that the anti-inflammatory effects of LBPs may be associated with the inhibition of NF-κB signaling.

An ELISA assay was used to investigate whether LBPs suppressed the release of TNF-α from LPS-stimulated BV2 cells. As shown in Fig. 3A, the 24-h LPS treatment of BV2 cells resulted in a marked increase in TNF-α levels compared with the controls. The application of LBPs significantly decreased the concentration of TNF-α in the culture media. Previous studies suggest that upon stress, HSP60 is upregulated and released into the extracellular space, where it exerts injury effects (21,22). We hypothesized that LPS also induces the increase of HSP60 in the extracellular space and this was confirmed by our experiment (Fig. 3B). LPS alone (200 ng/ml) markedly increased the HSP60 release compared with the controls. LBPs significantly reduced the HSP60 concentration in the medium. These results indicate that LBPs effectively suppress the production of TNF-α and HSP60 by over-activated microglia.