SK-N-SH human neuroblastoma cells (Type Culture Collection of the Chinese Academy of Sciences, Shanghai, China) were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% (v/v) fetal bovine serum, 100 U/ml penicillin/streptomycin (all from Invitrogen, Carlsbad, CA, USA) in plastic 25-cm² flasks at 37°C and under 5% CO₂, 95% air. Culture medium was changed every other day and the cells were subcultured once attaining 70–80% confluency. Morroniside (Phytomarker Ltd., Tianjin, China) was dissolved in D-PBS (Invitrogen) and H₂O₂ (Sinopharm Chemical Reagent Co. Ltd., Shanghai, China) was dissolved in sterile distilled water. SK-N-SH cells were pretreated with different concentrations of morroniside for 24 h, followed by incubation with H₂O₂ (200–400 µM) for 24 h to induce injury.

The viability of the cells was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Briefly, SK-N-SH cells were plated into 96-well plates at the density of 5×10³ cells/well. Cells were preincubated with morroniside (1, 10 and 100 µM) for 24 h before 200 µM H₂O₂ was administered. After a 24-h incubation, 20 µl of a 5 mg/ml stock solution of MTT (Sigma, St. Louis, MO, USA) was added to the culture medium and incubated in the dark for 4 h at 37°C to allow for formazan formation. Then MTT formazan crystals were dissolved in 100 µl dimethyl sulfoxide (DMSO; Sigma) and spectrophotometrically determined at an absorbance of 570 nm. The percentage of cell viability was measured by normalization of all values to the control group (=100%).

SK-N-SH cells were seeded into 6-well plates at the density of 2×10⁵ cells/well. When cell confluency achieved 70–80%, the cells were pre-incubated with morroniside for 24 h before H₂O₂ (200 µM) was added to induce cell damage for another 24 h. The growth and morphological changes in each group were observed under an inverted phase contrast microscope.

Lactate dehydrogenase (LDH) assay was used to assay H₂O₂-induced cytotoxicity. SK-N-SH cells were seed into 96-well plates at a density of 5×10³ cells/well. After cells were exposed to H₂O₂ (200 µM) for 24 h, a total of 120 µl of cell medium was collected for LDH analysis according to the manufacturer's protocol included in the cytotoxicity assay kit (Beyotime Biotechnology, Nantong, China). The absorbance of each group was read at 490 nm. Each group had five duplicate wells and the experiments were repeated at least three times. Data are expressed as the percentage of LDH release of the injury group (H₂O₂-induced group).

In order to determine whether morroniside protects against H₂O₂-induced apoptosis, Hoechst 33342 (Sigma), a fluorescent nuclear dye, was used to detect cell apoptosis by observing the cell morphology. Briefly, SK-N-SH cells were treated as described above and then washed with phosphate-buffered saline (PBS) and incubated with Hoechst 33342 (10 µg/ml in PBS; Sigma) for 15 min at 37°C in the dark. The cells were washed with PBS, fixed with cold 4% paraformaldehyde for another 15 min in room temperature, washed with PBS again, and then examined by fluroescence microscopy. The percentage of apoptotic cells was calculated as the ratio of apoptotic cells to the total cells counted. At least 500 cells were counted from more than 3 random microscopic fields.

Moreover, in order to confirm the anti-apoptotic effect of morroniside, flow cytometry with Annexin V-FITC and propidium iodide (PI) double staining (Beyotime Biotechnology) was performed. After SK-N-SH cells were treated with H₂O₂ (200 µM), 5×10⁵ cells were collected and counted for Annexin V-FITC and PI double staining. Briefly, cells in different groups were washed three times with cold PBS and stained with Annexin V-FITC for 15 min in the dark. After cells were washed another three times, PI was added and the fluorescence of each group was immediately analyzed by flow cytometry.

For visualization and analysis of intracellular ROS, the oxidation sensitive probe DCFH-DA (Beyotime Biotechnology) was used. After the treatment of H₂O₂, SK-N-SH cells were exposed to 10 µM DCFH-DA for 20 min at 37°C in the dark. The cells were washed with PBS for 3 times, and then DCF fluorescence was observed using fluorescence microscopy and quantified by fluorescence multi-well plate reader (BioTek, Highland Park, VT, USA) with an excitation wavelength of 488 nm and emission wavelength of 525 nm.

Superoxide anion was detected with dihydroethidium probes. Cells were treated with 2 µM dihydroethidium (Beyotime Biotechnology) for 30 min at 37°C in dim light after incubation with H₂O₂. Each well was washed with cold PBS and then DMEM to remove the remaining probes. Then cells were observed using fluorescence microscopy and the fluorescence intensity was quantified by a fluorescence multi-well plate reader.

Lipid peroxidation was monitored by measuring malondiadehyde (MDA; Beyotime Biotechnology), a stable end product of lipid peroxidation cascades using an MDA assay kit. SK-N-SH cells were washed with ice-cold PBS and then harvested with RIPA lysis buffer (Beyotime Biotechnology) after the treatment of H₂O₂. Cell homogenates were centrifuged at 16,000 × g at 4°C for 10 min. The supernatant was used for MDA assay and protein determination. The total protein concentrations were measured using BCA Protein assay kit (Beyotime Biotechnology). For MDA measurement, 100 µl samples were added into a 15-ml tube followed by addition of 200 µl MDA working solution. The mixture was heated at 100°C for 15 min, chilled to room temperature, and centrifuged at 1,000 × g for 10 min. Supernatants of 200 µl were transferred to 96-well plates, and the absorbance of each group was read at 532 nm.

Cellular SOD levels were determined using a Superoxide Dismutase assay kit (Jiancheng Bioengineering Institute, Nanjing, China). SK-N-SH cells were pretreated with different concentrations of morroniside for 24 h prior to exposure to H₂O₂ (200 µM) for another 24 h. Cells were washed twice with ice-cold PBS and harvested in RIPA lysis buffer, and then total protein contents were determined with the BCA protein assay kit (Beyotime Biotechnology). Samples were collected and analyzed according to the manufacturer's instructions. SOD levels were normalized to the protein concentrations.

SK-N-SH cells were treated with various dose of morroniside in a 12-well plate for 24 h before H₂O₂ (200 µM) for 24 h. A fluorescent dye JC-1 (Beyotime Biotechnology) was added to achieve final concentration of 10 µg/ml. Then cells were incubated for 30 min at 37°C in dim light. Cells were washed with PBS to remove the excess dye, and then images were captured using fluorescence microscopy within 30 min. For the JC-1 monomer, excitation wavelength was 514 nm and maximum emission wavelength was 529 nm. For the JC-1 polymer, the maximum excitation and emission wavelength were 585 and 590 nm, respectively.

Activity of caspase-3 was measured with a commercial caspase-3 activity assay kit (Beyotime Biotechnology), using Ac-DEVD-pNA as the specific substrate. The cell pellets were lysed on ice in lysis buffer for 15 min, and then the lysate was centrifuged at 20,000 × g for 10 min at 4°C. Supernatant of 10 µl was collected and incubated with 10 µl Ac-DEVD-pNA (2 mM) at 37°C for another 1–2 h in the dark. The absorbance values were measured with a spectrofluorometer at 405 nm and were corrected as protein content in the lysate. Protein concentration was determined by Bradford protein assay kit (Beyotime Biotechnology) according to the manufacturer's instructions.

The mRNA expression levels of B cell lymphoma-2 (Bcl-2) and Bcl-2-associated X protein (Bax) were determined by RT-PCR. Briefly, SK-N-SH cells were treated as mentioned above, and the total RNA of each group was extracted using TRIzol agent (Invitrogen) according to the manufacturer's instructions. Two micrograms of total RNA was first reverse transcribed into cDNA, and then routine PCR was performed as previously described (22). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal control and products were analyzed on 1% agarose gel. ImageJ software (NIH, Bethesda, MD, USA) was used to quantify the optical density value of each band. The sequences of specific primers (Sangon Biotech Inc., Shanghai, China) used for RT-PCR are listed in Table I.

Cells were collected at 24 h after exposure to H₂O₂. In order to detect the expression of Bcl-2 and Bax, SK-N-SH cells were washed with PBS and lysed using a lysis buffer for 30 min on ice. Lysates were centrifuged at 16,000 × g for 10 min at 4°C, and the supernatant was saved for the total protein determination. Protein concentrations were determined using a commercially available BCA protein assay kit (Beyotime Biotechnology). For western blot analysis, equivalent amounts of protein of each sample (20 µg) were electrophoresed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA). After the membranes were blocked with 5% non-fat dry milk in Tris-buffered saline (TBS) for 1 h at room temperature, primary antibodies (in TBST-5% BSA) against Bcl-2 (1:2,000; 12789-1-AP) and Bax (1:5,000; 60267-1-Ig) (both from ProteinTech Group Inc, Chicago, IL, USA) were added and incubated overnight at 4°C for determining signal transduction events. The membranes were rinsed three times with TBST and incubated with an appropriate HRP-conjugated secondary antibody (1:2,000; #04-15-06 and #04-18-06) (both from KPL, Gaithersburg, MD, USA) for 1 h at 37°C. An ECL kit (Millipore) was used to visualize membrane immunoreactivity. Quantification was performed using a computerized imaging program Quantity One (Bio-Rad, Hercules, CA, USA).

All data are presented as mean ± standard deviation of the mean (SD). Statistical analyses were performed by one-way analysis of variance (ANOVA) with post hoc Tukey's t-test to determine statistical significance. A value of P<0.05 was considered to indicated statistically significant differences.

H₂O₂-induced cell injury was evaluated using the MTT assay. H₂O₂ reduced cell viability in a dose- and time-dependent manner, with a survival rate of 50% after 24 h in the presence of 200 µM H₂O₂ relative to the untreated controls (Fig. 2A). Cells were treated with various concentrations of morroniside for 24 h to determine whether the H₂O₂-induced decrease in viability would be mitigated. Morroniside had no effect on the survival of SK-N-SH cells that were not treated with H₂O₂ (Fig. 2B). However, pretreatment with morroniside attenuated the H₂O₂-induced decrease in cell survival in a dose-dependent manner, with the greatest effect observed at 10 µM (Fig. 2C).

SK-N-SH cells treated for 24 h with 200 µM H₂O₂ showed morphological changes including loss of cell projections; a round, swollen, or shrunken cell body; and aggregation. These changes were blocked by pretreatment with morroniside (Fig. 2D).

The LDH release assay showed that H₂O₂ increased LDH activity in the supernatant of the SK-N-SH cell cultures relative to the control group. This effect was suppressed upon treatment with 1, 10 and 100 µM morroniside (P<0.05) (Fig. 2E).

We investigated whether the H₂O₂-induced decrease in SK-N-SH cell survival was due to apoptosis by Hoechst 33342 and Annexin V/PI staining. Control cells stained with Hoechst 33342 had large, oval-shaped nuclei (Fig. 3A). H₂O₂ (200 µM) treatment increased the fraction of cells with condensed or fragmented nuclei (18.60±1.67 vs. 2.00±1.00%; P<0.01) (Fig. 3B and F). These defects were rescued by pretreatment with morroniside (1, 10 and 100 µM), which reduced the fraction of apoptotic cells (14.80±0.84, 10.40±1.14 and 13.20±1.48%, respectively vs. 18.60±1.67%; P<0.01) (Fig. 3C–F). Consistent with these findings, there were few cells positive for Annexin V/PI staining detected in the control group (Fig. 3G). H₂O₂ (200 µM) treatment increased the percentage of Annexin V⁺/PI⁺ cells (34.58±4.59 vs. 4.96±2.44%; P<0.001) (Fig. 3H and L), which was reduced by morroniside (1, 10 and 100 µM) treatment (26.86±4.05, 22.42±2.81 and 22.62±2.48%, respectively vs. 34.58±4.59%; P<0.05) (Fig. 3I–L).

Intracellular ROS levels were detected with 2,7′-dichlorofluorescein (DCF) diacetate. SK-N-SH cells exposed to 200 and 400 µM H₂O₂ for 24 h showed dose-dependent increases in intracellular DCF fluorescence (P<0.01) (Fig. 4A–C and G), indicating that ROS levels were elevated. However, 10 µM morroniside pretreatment abrogated these increases in fluorescence intensity compared to the H₂O₂-treated groups (P<0.01) (Fig. 4E–G), suggesting a protective effect against H₂O₂-induced free radical formation.

Superoxide anion is a type of ROS that is associated with oxidative stress. To determine whether it is involved in H₂O₂-induced cytotoxicity, we measured intracellular superoxide anion levels with the dihydroethidium probe (Fig. 4H–M). The fluorescence intensity of SK-N-SH cells increased markedly after incubation for 24 h with H₂O₂ (P<0.001) (Fig. 4I and M). However, pretreatment with 1, 10 and 100 µM morroniside significantly reduced intracellular superoxide anion levels.

Lipid peroxidation levels were monitored by detecting the stable end products of lipid peroxidation using an MDA assay kit. Under normal conditions, cellular MDA content was ~119.47±14.14 nmol/g protein (Fig. 5). In cells treated with 200 µM H₂O₂ for 24 h, the level was increased to 296.28±24.25 nmol/g protein (P<0.001). However, pretreatment with 1, 10 and 100 µM morroniside reduced MDA levels to 256.26±6.70, 223.63±12.47 and 224.35±10.18 nmol/g protein, respectively (P<0.05).

To investigate the antioxidant properties of morroniside, we measured intracellular levels of SOD. Treatment with 200 µM H₂O₂ for 24 h reduced intracellular SOD levels from 6.77±0.45 to 3.74±0.41 U/mg (P<0.001). This was reversed by morroniside pretreatment at concentrations ranging from 1 to 100 µM; for example, a concentration of 10 µM enhanced SOD level by over 45% relative to the H₂O₂-treated group (Fig. 6).

We investigated whether morroniside could suppress the H₂O₂-induced decrease in MMP using JC-1 dye. SK-N-SH cells treated with 200 µM H₂O₂ for 24 h showed an obvious decrease in MMP, as evidenced by a decrease in the ratio of red to green fluorescence (Fig. 7). This effect was mitigated by treatment with morroniside, most obviously at a concentration of 10 µM.

Caspase-3 is a key protein in the regulation of apoptosis. SK-N-SH cells treated with 200 µM H₂O₂ for 24 h showed a nearly 2-fold increase in the caspase-3 level relative to the control cells (P<0.01) (Fig. 8). Pretreatment with morroniside (1, 10 and 100 µM) suppressed the H₂O₂-induced upregulation of caspase-3 (3.77±0.31, 3.05±0.16 and 3.31±0.12 µM pNA/g, respectively vs. 4.91±0.36 µM pNA/g; P<0.05).

The expression levels of the apoptosis-related genes Bcl-2 and Bax were altered in the SK-N-SH cells by H₂O₂ administration; the Bcl-2 mRNA level was downregulated while that of Bax was upregulated (Fig. 9A–C). These changes were abolished by pretreatment with 1–100 µM morroniside. Similarly, western blot analysis revealed that H₂O₂ (200 µM) decreased the Bcl-2 level (P<0.001) and increased that of Bax (P<0.01) after 24 h (Fig. 9D–F). However, pretreatment with 1, 10 and 100 µM morroniside restored Bcl-2 and Bax expression levels compared to these levels in the H₂O₂-induced group.