Baicalein and baicalin (Figure 1) with purity > 98% were purchased from Shanghai Innovative Research Center of Traditional Chinese Medicine (SIRC/TCM). Stock solutions (100 mM) were prepared in DMSO and diluted with serum-free medium. Dulbecco's Modified Eagle Medium with Nutrient Mixture F-12 (DMEM/F-12), Fetal Bovine Serum (FBS) and Penicillin-Streptomycin were purchased from GIBCO BRL (Grand Island, NY, USA). 2,7-Dichlorofluorescein diacetate (DCFH-DA) and Rhodanmine 123 (Rh123) were purchased from Molecular Probes (Invitrogen, CA, USA). Rotenone, Hoechst 33258, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), RIPA buffer, BCA Protein Assay Kit and other chemicals were obtained from Sigma-Aldrich (St. Louis, MO, USA). PVDF membrane was purchased from Millipore (MA, USA). Primary antibodies against Bax (D21), Bcl-2 (C21), β-actin and horseradish peroxidase (HRP)-conjugated secondary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Primary antibodies against phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) and cleaved caspase-3 (Asp175) were purchased from Cell Signaling (Beverly, MA, USA). ECL™ Western blotting detection system was purchased from Amersham Biosciences (Piscataway, NJ, USA).

Human neuroblastoma SH-SY5Y cells (passage ≤ 25) were cultured as described in our previous study [²¹] and then treated with different concentrations of rotenone, baicalein or baicalin respectively in serum-free medium for 24 hours to determine their cytotoxicity. To evaluate the protective effects, we pretreated SH-SY5Y cells with different concentrations of baicalein or baicalin for 1 hour and subsequently rotenone was added to the cells for another 24 hours. The final concentration of DMSO in the medium was 0.5%, and showed no cytotoxicity to the cells.

SH-SY5Y cells seeded on 96-well plates at 80-90% confluence were used in the MTT assay as described in our previous study [²¹]. In brief, the medium was removed after the treatment. MTT solution (50 μl, 0.5 mg/ml in DMEM/F12) was added to each well and incubated for 4 hours at 37°C. MTT lysis buffer containing 50 μl of 20% SDS (sodium dodecyl sulfate), 50% DMF (N, N-dimethylformamide), adjusted to pH4.7 by HCl (hydrogen chloride) was then added before overnight incubation of the cells at 37°C to dissolve formazan. The absorbance at 570 nm was measured by a microplate reader (Model 680, Bio-Rad Laboratories, UK). The cell viability was expressed as percentage of the control.

SH-SY5Y cells were incubated with different concentrations of baicalein in serum-free medium for 1 hour, followed by the co-treatment with rotenone (20 μM) for another 24 hours. Chromosomal DNA was stained with a fluorescent DNA-binding probe Hoechst 33258 (5 μg/ml) for 5 minutes, washed with PBS and then observed by a Axiovert S-100 Zeiss fluorescent microscope (Carl Zeiss, Zürich, Switzerland) at 20×. The morphological changes were visualized by phase-contrast imaging at 20×.

SH-SY5Y cells were pretreated with different concentrations of baicalein for 1 hour and then co-treated with rotenone (20 μM) for another 6 hours in serum-free medium. According to the protocols described in our previous study [²¹], the fluorescent probes DCFH-DA and Rh123 were used to determine the generation of intracellular ROS and mitochondrial membrane potential (ΔΨm), respectively. The total cell counts and fluorescent intensity were calculated with Image J software (ImageJ 1.45, http://rsbweb.nih.gov/ij). Mean fluorescent intensity (MFI) was calculated for each group using the following formula:

SH-SY5Y cells were pre-incubated for 1 hour with different concentrations of baicalein and then co-treated with rotenone (20 μM) for another 24 hours in serum-free medium. Total proteins were extracted using RIPA buffer. Protein determination was by a BCA Protein Assay Kit. Denatured proteins (30 μg) were size fractionated by 12.5% SDS-polyacrylamide gels. Proteins were transferred to PVDF membrane at 80 V for 3 hours. The blots were blocked for 1 hour at room temperature in fresh blocking buffer (0.1% Tween-20 in Tris-buffered saline, pH7.4, containing 5% BSA). The membrane was incubated overnight at 4°C with primary antibodies against Bax, Bcl-2, cleaved caspase-3 and phosphorylated ERK1/2 at dilution of 1:1000. β-actin was used as a loading control. The membrane was incubated for 2 hours with HRP-conjugated secondary antibodies at a dilution of 1:2000. Signals were detected using ECL™ Western blotting detection system. Protein bands were semi-quantified by densitometric analysis using Image J software.

Each experiment was performed at least three times, and the results were presented as means or means ± standard deviations (SD). One-way analysis of variance (ANOVA) followed by Student-Newman-Keuls test for multiple comparison was performed using the SigmaPlot 11.0 software packages (Systat Software Inc., San Jose, CA, USA). Exact P values were unavailable due to the software features (Additional file 1 provides a screen snapshot for example). Dose dependence was visually determined from the dose-response graphs. A probability value of P < 0.05 was considered to be statistically significant.

The cytotoxicity of rotenone, baicalein and baicalin were determined by MTT assay, Figure 2A shows that the cell viability was decreased in a dose-dependent manner (P < 0.01) by the treatment with rotenone for 24 hours. Rotenone (20 μM) triggered about 50% cell death and this concentration was chosen for subsequent experiments. Both baicalein and baicalin showed no cytotoxicity at the concentrations ranged 10-100 μM. Figure 2B shows that baicalein increased cell viability by 20-40% (P < 0.01) at all the tested concentrations, compared with the control.

The effect of baicalein and baicalin on rotenone-induced cell death was evaluated. Figures 2C-D show that pre- and subsequent co-treatment of baicalein significantly inhibited rotenone-induced cell death in a dose-dependent manner (P < 0.01). Baicalein (25-100 μM) increased the cell viability up to or even more than the control level (P < 0.01). In consistency with the MTT result, the morphological observations revealed that baicalein significantly reversed the cellular damage triggered by rotenone, as shown in Figure 2E. However, baicalin showed no statistically significant protective effect against rotenone-induced cell death.

Compared with the control, the apoptotic characteristics induced by the rotenone treatment, such as nuclear condensation and fragmentation, could be attenuated by pre- and subsequent co-treatment with increasing concentrations of baicalein (as shown in Figure 3). The statistical data showed 4.29 ± 0.69 folds of increase in the ratio of apoptotic cells triggered by rotenone, which could be reduced to the control level by pre- and subsequent co-treatment with increasing concentrations of baicalein (P < 0.01). Baicalein treatment for 24 hours had no significantly effect on nuclear apoptosis.

Figure 4 demonstrates that rotenone treatment induced 2.19 ± 0.36 folds of increase in the intracellular ROS compared with the control (P < 0.01). Pre- and subsequent co-treatment with baicalein reduced the production of ROS in a dose-dependent manner (P < 0.01) down to the control level. Baicalein treatment for 6 hours showed no significant effect on ROS production as compared with the control.

The inhibition of complex I by rotenone may induce loss of ΔΨm and the release of pro-apoptotic proteins [²³]. As shown in Figure 5, rotenone treatment led to about 2 folds of decrease in Rh123 fluorescence (P < 0.01), reflecting the loss of ΔΨm. Pre- and subsequent co-treatment with baicalein significantly inhibited the loss of ΔΨm in a dose-dependent manner (P < 0.01). Baicalein treatment for 6 hours showed no significant effect on ΔΨm as compared with the control.

To further characterize the mechanism of baicalein inhibition on rotenone-induced apoptosis, we determined the effect of baicalein on the expression of anti- and pro-apoptotic proteins by Western blots. As shown in Figure 6, the expression of Bax and cleaved caspase-3 was increased while the expression of Bcl-2 was significantly decreased by the treatment with rotenone (20 μM) for 24 hours (P < 0.05), compared with the control. Pre- and subsequent co-treatment with increasing concentrations of baicalein gradually restored the imbalanced expression profile of these proteins. Interestingly, baicalein treatment alone for 24 hours could reduce the base levels of Bax (0.86 ± 0.07) and cleaved caspase-3 (0.71 ± 0.09) (P < 0.05).

It was reported that rotenone induced ERK1/2 phosphorylation and neuronal degeneration in hippocampus neurons [²⁴]. Similar to this finding, we detected 2.47 ± 0.18 folds of increase in the expression of phosphorylated ERK1/2 in SH-SY5Y cells by treatment with rotenone for 24 hours, as shown in Figure 6 (P < 0.05). Pre- and subsequent co-treatment with baicalein reduced the expression of phosphorylated ERK1/2 down to the control level in a dose-dependent manner. Baicalein treatment alone for 24 hours could also significantly reduce the base level of ERK1/2 phosphorylation.