GW was obtained from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Zinc protoporphyrin-IX (Znpp-IX, an HO-1 inhibitor), dimethyl sulfoxide (DMSO), trypsin and MTT were from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). All culture reagents were from Invitrogen (Thermo Fisher Scientific, Inc., Waltham, MA, USA). LPS, phenylmethanesulfonyl fluoride (PMSF), radioimmunoprecipitation assay (RIPA) buffer, the enhanced bicinchoninic acid (BCA) protein assay kit, all SDS-PAGE reagents, the nuclear and cytoplasmic protein extraction kit, the enhanced chemiluminescence (ECL) substrate reagent kit and the caspase-3 activity kit were obtained from Beyotime Institute of Biotechnology (Jiangsu, China). The Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) detection kit was from KeyGen Biology (Nanjing, China). Rabbit polyclonal antibodies specific for cleaved caspase-3 (CC3; cat. no. 9661), apoptosis regulator Bcl-2 (bcl-2; cat. no. 2876), apoptosis regulator BAX (bax; cat. no. 2772), NF-κB p65 (cat. no. 3034) and anti-GAPDH (cat. no. 2118; 1:1,000) were from Cell Signaling Technology, Inc. (Danvers, MA, USA). Rabbit polyclonal antibodies specific for HO-1 (cat. no. SPA-896) and histone 3 (H3; cat. no. ab1791) were obtained from Stressgen Bioreagents Corporation (Victoria, BC, Canada) and Abcam (Cambridge, UK), respectively.

H9c2 cardiomyoblasts (CRL-1446; American Type Culture Collection, Manassas, VA, USA) were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 U/ml penicillin and 100 µg/ml streptomycin in a humidified atmosphere of 5% CO₂ and 95% air at 37°C. Cells were passaged regularly and subcultured to 90% confluence prior to experimental procedures.

GW dissolved in DMSO was diluted with low-serum medium (1% FBS/DMEM) to the final concentrations (25, 50 and 100 nmol/l) before use. The final concentration of DMSO in the incubation mixture was not more than 0.1% (v/v). Prior to experimental intervention, confluent cultured cells were serum-starved for 24 h in low-serum medium. Cells were pretreated with different doses of GW for 24 hat 37°C, and then incubated with 1 µg/ml of LPS for 24 h. For the inhibitor experiment, cells were pretreated with 100 nM of GW for 24 h at 37°C in the absence or presence of 10 µmol/l of ZnPP-IX, which was added 1 h before GW, and then the cells were incubated for 24 h with 1 µg/ml LPS.

Cell viability was assessed by MTT assay. Following incubation with the test reagents, 5 mg/ml MTT was added to the culture media and the cells were incubated for an additional 4 h. Subsequent to this incubation, the resulting formazan was solubilized by adding DMSO, and the optical density of the solubilized cell extract was measured at 490 nm using a microplate reader.

Apoptosis was detected using an Annexin V-FITC/PI detection kit according to the manufacturer's protocol. The cells were digested with 0.25% trypsin, washed with ice-cold PBS and resuspended in binding buffer (5×10⁵ cells/ml). The cells were centrifuged at 1,000 × g for 5 min at 4°C. Subsequent to the supernatant being discarded, 500 µl binding buffer, 5 µl Annexin V-FITC and 5 µl PI were added to the cell suspension. Following gentle mixing, the suspensions were incubated for 15 min at room temperature without light. The cells were analyzed by flow cytometry (BD LSRII; BD Biosciences, Franklin Lakes, NJ, USA). Data analysis was performed using CellQuest version 3.3 (BD Biosciences).

Caspase-3 activity was determined using a caspase-3 activity kit, which detects the production of the chromophore p-nitroanilide following its cleavage from the peptide substrate DEVD-p-nitroanilide. According to the manufacturer's protocol, following washing with cold PBS, cells were lysed with lysis buffer (100 µl/2×10⁶ cells) for 15 min on ice. The mixture, composed of 10 µl cell lysate, 80 µl reaction buffer and 10 µl 2 mmol/l caspase-3 substrate, was incubated in 96-well plates at 37°C for 4 h, and caspase-3 activity was quantified in the samples using a microplate reader at an absorbance of 405 nm.

Cells from each group were washed twice with ice-cold PBS and lysed in RIPA buffer (10 mmol/l Tris, pH 7.4; 150 mmol/l NaCl; 1 mmol/l EDTA; 0.1% SDS; 1% Triton X-100; and 1% sodium deoxycholate) with protease inhibitors (1 mmol/l PMSF, 1 µg/ml aprotinin, 1 µg/ml pepstatin, and 1 µg/ml leupeptin) at 4°C. The lysate was centrifuged at 12,000 × g at 4°C for 20 min to remove the insoluble material. Supernatants were collected. The protein concentration was determined using the BCA assay. Equal quantities of protein (30 µg) were separated by 10% SDS-PAGE, and subsequently transferred onto a polyvinylidene difluoride (PVDF) membrane. The membranes were blocked in TBS with Tween-20 (TBST) with 5% (w/v) skimmed milk at room temperature for 2 h, followed by overnight incubation at 4°C with primary antibodies diluted in 0.1% TBST. Following washing in TBST, the membranes were incubated at room temperature for 1 h with goat anti-rabbit horseradish peroxidase-conjugated secondary antibody (cat. no. 7074; 1:3,000; Cell Signaling Technology, Inc.) diluted in 0.1% TBST. Following washing once more in TBST, the immunoreactive bands were visualized by using an ECL kit and exposed to X-ray film. The band intensity was quantified using Quantity One software (Bio-Rad Laboratories Inc. Hercules, CA, USA).

The extraction and isolation of nuclear proteins was performed using the nuclear and cytoplasmic protein extraction kit according to the manufacturer's protocol. Cells were detached with cold PBS and centrifuged at 1,000 × g for 5 min at 4°C, and the pellet was dissolved with cytoplasmic protein extraction agent A supplemented with PMSF. Subsequent to mixing by vortex for 5 sec, the tubes were incubated for 10–15 min on ice to promote lysis. Cytoplasmic protein extraction agent B was added and mixing by vortex was performed for 5 sec, followed by incubation on ice for 60 sec. The samples were centrifuged for 5 min at 14,000 × g at 4°C and the supernatant, consisting of the cytosolic fraction, was immediately frozen for further analysis. The pellet was re-suspended in nuclear protein extraction agent supplemented with PMSF. Subsequently, the tubes were mixed by vortex 15–20 times over the course of 30 min and centrifuged for 10 min at 14,000 × g at 4°C to obtain supernatants containing the nuclear extracts. Protein concentration was measured using the BCA protein assay kit. Levels of NF-κB p65 in the nuclear protein extract were determined by western blot analysis as described above.

All data represent the mean of samples from three separately-performed experiments. Results are expressed as mean ± standard deviation. One-way analysis of variance and Student-Newman-Keuls tests were used for statistical evaluation with SPSS 14.0 software (SPSS, Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.

Using the MTT assay, a possible cytoprotective effect of GW against LPS-induced cell death was detected. To evaluate whether GW was cytotoxic to H9c2 cells, the cells were first treated with concentrations of GW ranging between 25 and 100 nmol/l for 24 h. A reduction in cell viability was not observed (Fig. 1A). Subsequent analyses tested whether GW was able to protect against LPS-induced cytotoxicity. When cells were pretreated for 24 h with growing concentrations of GW (25–100 nmol/l), prior to treatment with 1 µg/ml LPS for 24 h, significant increases in cell viability were observed compared with treatment with LPS alone. As exhibited in Fig. 1B, pretreatment with 50 and 100 nmol/l GW significantly increased cell viability to 70.89±5.61 and 81.16±7.12 % of the control, respectively, compared with treatment with LPS alone. These results indicate that GW may have a protective role against LPS-induced cell cytotoxicity.

Apoptosis was quantitated by flow cytometry using Annexin V-FITC/PI staining. As presented in Fig. 2, LPS led to a significant increase in the apoptotic rate of H9c2 cells compared with the control group, and the apoptotic rate was decreased markedly by pretreatment with GW compared with the LPS alone group. Subsequent western blot analysis demonstrated that CC3 protein expression was markedly increased in LPS-stimulated cells compared with the control group, while GW pretreatment decreased CC3 protein expression in a dose-dependent manner compared with the LPS group (Fig. 3). Western blot analysis also indicated that GW notably inhibited the decrease of the bcl-2/bax ratio induced by LPS in a dose-dependent manner (Fig. 4).

To investigate the molecular mechanisms by which GW protects H9c2 cells from LPS-induced apoptosis, the nuclear NF-κB protein expression level was examined to determine whether the regulation of NF-κB is responsible for the anti-apoptotic effect of GW. As displayed in Fig. 5, when cells were pretreated for 24 h with increasing concentrations of GW prior to incubation with LPS, NF-κB protein expression was decreased in a dose-dependent manner compared with the LPS group. These results suggest that GW prevents LPS-induced apoptosis, possibly through inhibition of NF-κB activation.

The cells were treated with increasing concentrations of GW (25–100 nmol/l). Treatment with GW for 24 h increased HO-1 expression in a dose-dependent manner (Fig. 6A). HO-1 expression was subsequently determined by treating the cells with 100 nmol/l GW for 12, 24 and 48 h. As presented in Fig. 6B, a maximum increase of HO-1 protein expression was observed following treatment for 24 h.

In order to validate the protective role of HO-1, H9c2 cells exposed to LPS were pretreated with 100 nmol/l GW for 24 h in the absence or the presence of 10 µM ZnPP-IX. As exhibited in Fig. 7, the caspase-3 activity was increased by 9.42±0.61-fold that of the control in the LPS group, whereas it was reduced to 3.07±0.24-fold that of the control following pretreatment with GW. Co-incubation with ZnPP-IX, which partly negated the effect of GW, increased the caspase-3 activity to 6.91±0.40-fold that of the control. These results suggest that the protective effect of GW on LPS-induced cell apoptosis is HO-1-dependent.

To determine whether the upregulation of HO-1 by GW is important to the survival of H9c2 cells, due to the modulation of NF-κB, the effects of ZnPP-IX on the expression of NF-κB were tested. H9c2 cells exposed to LPS were pretreated with 100 nmol/l GW for 24 h in the absence or the presence of 10 µM ZnPP-IX. As presented in Fig. 8, ZnPP-IX co-incubation reversed the inhibitory effect of GW on LPS-induced NF-κB activation, suggesting that HO-1 may mediate the inhibitory effect of GW on NF-κB activation in LPS-stimulated H9c2 cells.