RESEARCH Open Access

Baicalein antagonizes rotenone-inducedapoptosis in dopaminergic SH-SY5Y cells relatedto ParkinsonismJu-Xian Song1, Mandy Yuen-Man Choi1, Kavin Chun-Kit Wong1, Winkie Wing-Yan Chung1, Stephen Cho-Wing Sze1,Tzi-Bun Ng2 and Kalin Yan-Bo Zhang1*

Abstract

Background: Two active compounds, baicalein and its glycoside baicalin were found in the dried root ofScutellaria baicalensis Georgi, and reported to be neuroprotective in vitro and in vivo. This study aims to evaluatethe protective effects of baicalein on the rotenone-induced apoptosis in dopaminergic SH-SY5Y cells related toparkinsonism.

Methods: Cell viability and cytotoxicity were determined by MTT assay. The degree of nuclear apoptosis wasevaluated with a fluorescent DNA-binding probe Hoechst 33258. The production of reactive oxidative species (ROS)and loss of mitochondrial membrane potential (m) were determined by fluorescent staining with DCFH-DA andRhodanmine 123, respectively. The expression of Bax, Bcl-2, cleaved caspase-3 and phosphorylated ERK1/2 wasdetermined by the Western blots.

Results: Baicalein significantly increased viability and decreased rotenone-induced death of SH-SY5Y cells in a dose-dependent manner. Pre- and subsequent co-treatment with baicalein preserved the cell morphology and attenuatedthe nuclear apoptotic characteristics triggered by rotenone. Baicalein antagonized rotenone-induced overproductionof ROS, loss of m, the increased expression of Bax, cleaved caspase-3 and phosphorylated ERK1/2 and thedecreased expression of Bcl-2.

Conclusion: The antioxidative effect, mitochondrial protection and modulation of anti-and pro-apoptotic proteinsare related to the neuroprotective effects of baicalein against rotenone induced cell death in SH-SY5Y cells.

BackgroundParkinsons disease (PD) is a neurodegenerative diseasemainly characterized by loss of dopaminergic neurons inthe substantia nigra pars compacta [1]. Although thepathology of PD is not understood well, the neurotoxicanimal models of PD represent some key neurobeha-vioral or pathological features [2]. Three neurotoxins, 6-hydroxydopamine (6-OHDA), 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and rotenone, are theagents to induce parkinsonism in vitro and in vivo [3].An extensive study of these models defined importantcellular actions of cell death and offered a basis for the

development of novel therapeutic strategies [4]. Rote-none, a lipophilic pesticide, can cross cell membraneeasily to induce systemic inhibition of mitochondrialcomplex I and cause selective nigrostriatal dopaminergicdegeneration [5]. Rotenone-induced apoptosis in humanneuroblastoma SH-SY5Y cells was mediated by the gen-eration of mitochondrial reactive oxygen species (ROS)[6].The rotenone model of PD has been used for identifying

potential neuroprotective agents in recent years [7]. Thismodel would enable scientific re-evaluation of various her-bals for treating PD [8] and facilitate the development ofnovel anti-parkinsonian drugs [9]. Baicalein and its corre-sponding glycoside baicalin are two flavonoid compoundsfound in the dried root of Scutellaria baicalensis Georgi.A series of studies demonstrated the neuroprotective

* Correspondence: zhang.yanbo@yahoo.com1School of Chinese Medicine, The University of Hong Kong, 10 SassoonRoad, Pokfulam, Hong Kong SAR, ChinaFull list of author information is available at the end of the article

Song et al. Chinese Medicine 2012, 7:1http://www.cmjournal.org/content/7/1/1

2012 Song et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative CommonsAttribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction inany medium, provided the original work is properly cited.

effects of baicalein or baicalin in experimental models ofAlzheimers disease [10,11], ischemic stroke [12-15] andPD [16-19]. Baicalein was reported to be effective on 6-OHDA models [18,20,21] and MPTP models [19,22] ofPD. This study aims to investigate the neuroprotectiveeffects of baicalein or baicalin on rotenone-induced cellu-lar toxicities (SH-SY5Y cells) in vitro and in vivo.

MethodsMaterialsBaicalein and baicalin (Figure 1) with purity > 98% werepurchased from Shanghai Innovative Research Center ofTraditional Chinese Medicine (SIRC/TCM). Stock solu-tions (100 mM) were prepared in DMSO and diluted withserum-free medium. Dulbeccos Modified Eagle Mediumwith Nutrient Mixture F-12 (DMEM/F-12), Fetal BovineSerum (FBS) and Penicillin-Streptomycin were purchasedfrom GIBCO BRL (Grand Island, NY, USA). 2,7-Dichloro-fluorescein diacetate (DCFH-DA) and Rhodanmine 123(Rh123) were purchased from Molecular Probes (Invitro-gen, CA, USA). Rotenone, Hoechst 33258, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT), RIPA buffer, BCA Protein Assay Kit and otherchemicals were obtained from Sigma-Aldrich (St. Louis,MO, USA). PVDF membrane was purchased from Milli-pore (MA, USA). Primary antibodies against Bax (D21),Bcl-2 (C21), b-actin and horseradish peroxidase (HRP)-conjugated secondary antibodies were purchased fromSanta Cruz Biotechnology (Santa Cruz, CA, USA). Primaryantibodies against phospho-p44/42 MAPK (ERK1/2)(Thr202/Tyr204) and cleaved caspase-3 (Asp175) werepurchased from Cell Signaling (Beverly, MA, USA). ECLWestern blotting detection system was purchased fromAmersham Biosciences (Piscataway, NJ, USA).

Cell culture and treatmentsHuman neuroblastoma SH-SY5Y cells (passage 25)were cultured as described in our previous study [21]and then treated with different concentrations of rote-none, baicalein or baicalin respectively in serum-freemedium for 24 hours to determine their cytotoxicity. To

evaluate the protective effects, we pretreated SH-SY5Ycells with different concentrations of baicalein or baica-lin for 1 hour and subsequently rotenone was added tothe cells for another 24 hours. The final concentrationof DMSO in the medium was 0.5%, and showed nocytotoxicity to the cells.

MTT assaySH-SY5Y cells seeded on 96-well plates at 80-90% conflu-ence were used in the MTT assay as described in our pre-vious study [21]. In brief, the medium was removed afterthe treatment. MTT solution (50 l, 0.5 mg/ml inDMEM/F12) was added to each well and incubated for4 hours at 37C. MTT lysis buffer containing 50 l of 20%SDS (sodium dodecyl sulfate), 50% DMF (N, N-dimethyl-formamide), adjusted to pH4.7 by HCl (hydrogen chloride)was then added before overnight incubation of the cells at37C to dissolve formazan. The absorbance at 570 nm wasmeasured by a microplate reader (Model 680, Bio-RadLaboratories, UK). The cell viability was expressed as per-centage of the control.

Cellular morphology and nuclear apoptosisSH-SY5Y cells were incubated with different concentra-tions of baicalein in serum-free medium for 1 hour, fol-lowed by the co-treatment with rotenone (20 M) foranother 24 hours. Chromosomal DNA was stained with afluorescent DNA-binding probe Hoechst 33258 (5 g/ml)for 5 minutes, washed with PBS and then observed by aAxiovert S-100 Zeiss fluorescent microscope (Carl Zeiss,Zrich, Switzerland) at 20. The morphological changeswere visualized by phase-contrast imaging at 20.

ROS and mitochondrial membrane potentialSH-SY5Y cells were pretreated with different concentra-tions of baicalein for 1 hour and then co-treated with rote-none (20 M) for another 6 hours in serum-free medium.According to the protocols described in our previousstudy [21], the fluorescent probes DCFH-DA and Rh123were used to determine the generation of intracellularROS and mitochondrial membrane potential (m),respectively. The total cell counts and fluorescent intensitywere calculated with Image J software (ImageJ 1.45, http://rsbweb.nih.gov/ij). Mean fluorescent intensity (MFI) wascalculated for each group using the following formula:

MFI = total uorescent intensity 100/total cell counts

Western blots analysisSH-SY5Y cells were pre-incubated for 1 hour with dif-ferent concentrations of baicalein and then co-treatedwith rotenone (20 M) for another 24 hours in serum-free medium. Total proteins were extracted using RIPA

Figure 1 The chemical structure of (A) baicalein and (B)baicalin.

Song et al. Chinese Medicine 2012, 7:1http://www.cmjournal.org/content/7/1/1

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buffer. Protein determination was by a BCA ProteinAssay Kit. Denatured proteins (30 g) were size fractio-nated by 12.5% SDS-polyacrylamide gels. Proteins weretransferred to PVDF membrane at 80 V for 3 hours.The blots were blocked for 1 hour at room temperaturein fresh blocking buffer (0.1% Tween-20 in Tris-bufferedsaline, pH7.4, containing 5% BSA). The membrane wasincubated overnight at 4C with primary antibodiesagainst Bax, Bcl-2, cleaved caspase-3 and phosphorylatedERK1/2 at dilution of 1:1000. b-actin was used as aloading control. The membrane was incubated for2 hours with HRP-conjugated secondary antibodies at adilution of 1:2000. Signals were detected using ECLWestern blotting detection system. Protein bands weresemi-quantified by densitometric analysis using Image Jsoftware.

Statistical analysisEach experiment was performed at least three times, andthe results were presented as means or means standarddeviations (SD). One-way analysis of variance (ANOVA)followed by Student-Newman-Keuls test for multiple com-parison was performed using the SigmaPlot 11.0 softwarepackages (Systat Software Inc., San Jose, CA, USA). ExactP 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 con-sidered to be statistically significant.

ResultsIn this study, we evaluated the effects of baicalein and bai-calin on rotenone-induced cell death, nuclear apoptosis,production of intracellular ROS, loss of m, expressionsof Bax, Bcl-2 and caspase-3, and phosphorylation ofERK1/2 in SH-SY5Y cells.

Cell deathThe cytotoxicity of rotenone, baicalein and baicalin weredetermined by MTT assay, Figure 2A shows that the cellviability was decreased in a dose-dependent manner (P