Coptis chinensis Franch. exhibits neuroprotective properties against oxidative stress in human neuroblastoma cells

Journal of Ethnopharmacology 155 (2014) 607–615

Contents lists available at ScienceDirect

Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jep

Coptis chinensis Franch. exhibits neuroprotective properties against oxidative stress in human neuroblastoma cells Thomas Friedemann a,n, Benjamin Otto b, Kristin Klätschke b, Udo Schumacher c, Yi Tao d, Alexander Kai-Man Leung d, Thomas Efferth e, Sven Schröder a a

HanseMerkur Center for Traditional Chinese Medicine at the University Clinic Hamburg-Eppendorf, Martinistr. 52, Hamburg 20246, Germany Array Service Center, University Medical Center Hamburg-Eppendorf, Martinistr. 52, Hamburg 20246, Germany c Institute of Anatomy and Experimental Morphology, University Medical Center Hamburg-Eppendorf, Martinistr. 52, Hamburg 20246, Germany d School of Chinese Medicine, Hong Kong Baptist University, 7 Baptist University Road, Kowloon Tong, Hong Kong Special Administrative Region, China e Department of Pharmaceutical Biology, Institute of Pharmacy and Biochemistry, Johannes Gutenberg University, Staudinger Weg 5, 55128 Mainz, Germany b

art ic l e i nf o

a b s t r a c t

Article history: Received 10 February 2014 Received in revised form 29 April 2014 Accepted 3 June 2014 Available online 12 June 2014

Ethnopharmacological relevance: The dried rhizome of Coptis chinensis Franch. (family Ranunculaceae) is traditionally used in Chinese medicine for the treatment of inflammatory diseases and diabetes. Recent studies showed a variety of activities of Coptis chinensis Franch. alkaloids, including neuroprotective, neuroregenerative, anti-diabetic, anti-oxidative and anti-inflammatory effects. However, there is no report on the neuroprotective effect of Coptis chinensis Franch. watery extract against tert-butylhydroperoxide (t-BOOH) induced oxidative damage. The aim of the study is to investigate neuroprotective properties of Coptis chinensis Franch. rhizome watery extract (CRE) and to evaluate its potential mechanism of action. Materials and methods: Neuroprotective properties on t-BOOH induced oxidative stress were investigated in SH-SY5Y human neuroblastoma cells. Cells were pretreated with CRE for 2 h or 24 h followed by 2 h of treatment with t-BOOH. To evaluate the neuroprotective effect of CRE, cell viability, cellular reactive oxygen species (ROS), mitochondrial membrane potential (MMP) and the apoptotic rate were determined and microarray analyses, as well as qRT-PCR analyses were conducted. Results: Two hours of exposure to 100 mM t-BOOH resulted in a significant reduction of cell viability, increased apoptotic rate, declined mitochondrial membrane potential (MMP) and increased ROS production. Reduction of cell viability, increased apoptotic rate and declined mitochondrial membrane potential (MMP) could be significantly reduced in cells pretreated with CRE (100 mg/ml) for 2 h or 24 h ahead of t-BOOH exposure with the greatest effect after 24 h of pretreatment; however ROS production was not changed significantly. Furthermore, microarray analyses revealed that the expressions of 2 genes; thioredoxin-interacting protein (TXNIP) and mitochondrially encoded NADH dehydrogenase 1, were significantly regulated. Down regulation of TXNIP was confirmed by qRT-PCR. Conclusion: Due to its neuroprotective properties CRE might be a potential therapeutic agent for the prevention or amelioration of diseases like diabetic neuropathy and neurodegenerative disorders like Alzheimer and Parkinsons disease. & 2014 Elsevier Ireland Ltd. All rights reserved.

Keywords: SH-SY5Y Oxidative stress Apoptosis Neuroprotection Coptidis rhizoma Thioredoxin-interacting protein

1. Introduction Diabetic neuropathy, neurodegenerative disease like Alzheimer or Parkinsons disease and aging are associated with oxidative

n

Corresponding author. Tel.: þ 49 40 42916376; fax: þ 49 40 42916349. E-mail addresses: [email protected] (T. Friedemann), [email protected] (B. Otto), [email protected] (K. Klätschke), [email protected] (U. Schumacher), [email protected] (Y. Tao), [email protected] (A.K.M. Leung), [email protected] (T. Efferth), [email protected] (S. Schröder). http://dx.doi.org/10.1016/j.jep.2014.06.004 0378-8741/& 2014 Elsevier Ireland Ltd. All rights reserved.

damage by reactive oxygen species (ROS) (Smith et al., 1996; Finkel and Holbrook, 2000; Perry et al., 2002; Vincent et al., 2004; Perfeito et al., 2012). Cells are usually protected from oxidative damage by the endogenous antioxidant system, which consists of thioredoxin, glutathione, ascorbic acid and a variety of enzymes like superoxide dismutase, catalase, thioredoxin reductase and glutathione reductase. If the antioxidative capacity of this system is exceeded or parts of the system are damaged, ROSinduced oxidative stress will occur. ROS accumulation can lead to damage of deoxyribonucleic acid, protein oxidation, membrane lipid peroxidation, depletion of cellular thiols and release of proinflammatory cytokines, which can in the end elicit tissue damage

608

T. Friedemann et al. / Journal of Ethnopharmacology 155 (2014) 607–615

and cell death (Gorman et al., 1996; Smith et al., 1996; Vincent et al., 2004). In a variety of studies the membrane permeant oxidant compound tert-butylhydroperoxide (t-BOOH) was used to induce oxidative stress (Amoroso et al., 1999; Amoroso et al., 2002; Zhao et al., 2005). Results showed that after t-BOOH permeates the cell membrane t-BOOH is modified to tert-butoxyl radicals by way of iron-dependent reactions and induces lipid peroxidation, DNA cleavage, depletion of intracellular glutathione, protein modifications, increase of intracellular Ca2 þ ions and as a consequence cell death (Rush and Alberts, 1986; Coleman et al., 1989; Guidarelli et al., 1997; Amoroso et al., 1999; Martin et al., 2001). Furthermore, it has been assumed that t-BOOH treatment leads to an opening of the mitochondrial permeability transition pore, which increases the permeability of the inner mitochondrial membrane and therefore leads to mitochondrial membrane potential depolarization (Nieminen et al., 1995, 1997; Zhao et al., 2005). By way of a downstream effect this can induce the release of cytochrome c (cyt c) into the cytosol (Kowaltowski et al., 2001). Cytosolic cyt c activates the caspase cascade leading to apoptotic cell death (Liu et al., 1996; Li et al., 1997). For this reason, therapeutic strategies targeting the prevention or attenuation of oxidative stress, DNA cleavage and mitochondrial membrane potential depolarization could have a major impact on the treatment of diseases associated with oxidative stress. Coptidis rhizoma, the dried rhizome of Coptis chinensis Franch. (Chinese goldthread, commonly known as wei-lian or huang-lian), is an herb used in Traditional Chinese Medicine (TCM) for inflammatory diseases like carbuncles, aphthae or ulcers as well as for dysentery, but ancient authors like Li Shizhen also used it to treat diabetes (Li, 1999). It is used in the treatment of various diseases in TCM due to its anti-diabetic, relaxant, pyretic, antibacterial, and antiviral effects (Huang, 1999). Some of its individual compounds showed a variety of activity forms, including neuroprotective (Luo et al., 2011), neuroregenerative (Han et al., 2012), anti-apoptotic (Miura et al., 1997), anti-oxidative (Gong et al., 2012), anti-inflammatory (Marinova et al., 2000) and anti-fungal effects (Seneviratne et al., 2008). The main components of Coptis chinensis Franch. are berberastine, berberine, columbamine, coptisine, epiberberine, jatrorrhizine and palmatine (Chuang et al., 1996; Wang et al., 2004; Zhao et al., 2010; Ding et al., 2012). In the traditional combination San Huang xie xin tang, which originated in the Ming dynasty, Coptis chinensis Franch. is combined with Rhei rhizoma and Scutellaria radix (Dong, 2000). This recipe has been shown to have neuroprotective capacities due to its antiinflammatory and antioxidative effects (Lo et al., 2012). However, there is no report on the neuroprotective effect of CRE against tBOOH induced oxidative damage. Therefore, we investigated the neuroprotective effect of CRE oxidative stress in SH-SY5Y human neuroblastoma cells by evaluation of the cell viability, ROS production, apoptotic rate, mitochondrial membrane potential and transcriptional changes.

CMX Ros were obtained from Life Technologies (Darmstadt, Germany) and dissolved in DMSO. Hoechst 33342 (Life Technologies) stock solution was prepared with 70% ethanol. All other reagents were purchased from Roth (Karlsruhe, Germany) or Sigma (Taufkirchen, Germany). 2.2. Herbal preparation The rhizome of Coptis chinensis Franch. was purchased as a dried herb from China Medica (Ch. B. 930034; 83684 Tegernsee, Germany), and tested for identity and purity by Sebastian Kneipp research laboratory for residue analysis and organic trace analysis (Bad Wörishofen, Germany). The plant name used in this publication was verified with www.theplantlist.org on February the 10th 2014. The rhizome of Coptis chinensis Franch. was ground into a fine powder and extracted twice for 30 min with boiling distilled deionized water (ratio: 1 g/10 ml). Supernatants were combined, concentrated with a rotary-vacuum evaporator (60 1C, 200 mbar; Rotavapor-R, Büchi) and dried to powder with a vacuum concentrator (Bachofer) at room temperature. 2.3. HPLC-analysis HPLC-analysis was conducted on an Agilent HPLC 1260 infinity (Agilent Technologies, Germany) using an Alltima C18 (250 mm  4.6 mm  5 mm, S/N: 213100139, temperature: 25 1C) column. The mobile phase consisted of 0.1% trifluoroacetic acid (A) and acetonitrile (B). Chromatographic separation was optimized according to the method described in the Hong Kong Chinese Materia Medica Standards (Department of Health of Hong Kong, 2008). The following gradient protocol was used with a flow rate of 1.0 ml/min: 0–30 min 20–50% B for separation and 31–40 min 90% B, 41–65 min 20% B for cleaning up and equilibrium. The column pressure at equilibrium was 115 bar. 346716 nm was used as detection wavelength and 610750 nm as the reference wavelength. Coptisine, palmatine and berberine (Cfm Oskar Tropitzsch; Markdredwitz; Germany) were used as reference standard compounds. HPLC-Data were analyzed with the Agilent ChemStation for LC (Rev. B.04.03, Agilent Technologies, Germany). 2.4. Free radical scavenging activity

2. Materials and methods

Antioxidant activity of CRE was determined by using the DPPH assay (Turkmen et al., 2006; Sharma and Bhat, 2009). The dried extract was dissolved in DDW to a final concentration of 56.25 mg/ml– 7.2 mg/ml and 50 ml was added to each well of a 96-well-plate. Afterwards, 200 ml of 75 mM DPPH was added to each well and the plate was agitated with 650 rpm for 5 min in the dark with an AIP 4 plate shaker (Diagnostics Pasteur). After a total incubation time of 30 min, absorbance was measured 3 times at 531 nm (Thermo Multiskan SPECTRUM microplate spectrophotometer). 50 ml DDW served as the negative control and trolox as well as N-acetyl-L-cystein (NAC) as the positive controls. Three independent experiments were conducted and each experiment was performed in triplicate.

2.1. Drugs

2.5. Peroxyl radical scavenging activity

2,2-Di(4-tert-octylphenyl)-1-picrylhydrazyl (DPPH; Aldrich, Taufkirchen, Germany) was dissolved in methanol. Sodium phosphate buffer (pH 7.4) was used to prepare fluorescein and 2,20 Azobis (2-methylpropionamidine) dihydrochlorid stock solution. Trolox (Sigma, Taufkirchen, Germany) was dissolved in 80% ethanol. Tert-butyl-hydroperoxide (t-BOOH; 48933, Fluka, Taufkirchen, Germany) working solution was prepared with RPMI 1640 medium. 20 ,70 -dichlorodihydrofluorescein diacetate, Mitotracker Red

The antioxidant capacity of CRE was determined by using the ORAC assay. The ORAC assay was carried out with slight modifications according to the method described by Gillespie et al. (2007). Briefly, 225 ml of 10 nM fluorescein solution dissolved in a 75 mM sodium phosphate buffer (SPB; pH 7.4) was pipetted into the well of a 96-well microplate and 37 ml of sodium phosphate buffer (blank), Trolox (20–80 mM) or the herbal extract (5–20 mg/ml; sample) was added in different concentrations. The microplate

T. Friedemann et al. / Journal of Ethnopharmacology 155 (2014) 607–615

was agitated for 30 min with 350 rpm at 37 1C. Afterwards, 37 ml 240 mM 2,20 -Azobis (2-methylpropionamidine)dihydrochlorid (AAPH) was added with a multichannel pipette to each well and the kinetic reading started immediately. For the fluorescence kinetic reading, a multi-detection plate reader (Mithras LB 940; Berthold) was used with an excitation wavelength of 485 nm and an emission wavelength of 528 nm. The fluorescent signal was measured every minute for 120 min.

2.6. Cell culture The human neuroblastoma SH-SY5Y cell line was established in 1970 and has been extensively used in the last decades as a neuronal model to study diabetic neuropathy and neurodegenerative disease like Alzheimers and Parkinsons disease (Hattangady and Rajadhyaksha, 2009; Xie et al., 2010; Gu et al., 2013). SH-SY5Y cells were cultivated in RPMI 1640 medium supplemented with 10% fetal calf serum, 100 U/ml penicillin and 100 mg/ml streptomycin in a humid atmosphere of 5% CO2 and 95% air at 37 1C.

2.7. Cell viability measurements Cell viability was determined by the thiazolyl blue tetrazolium bromide (MTT) assay. This assay is based on the conversion of dissolved yellow MTT by the mitochondrial succinate dehydrogenase of living cells into insoluble purple formazan. The amount of formazan generated by the cells reflects the mitochondrial metabolic capacity and the intracellular redox state (Zeng et al., 2004). Therefore, it is a measure of cell viability and mitochondrial activity. Briefly, 4  104 cells were seeded into the well of a 96-well microtitre plate and incubated for 24 h in a humid atmosphere of 5% CO2 and 95% air at 37 1C. The dried CRE was dissolved in medium and sterilely filtrated. Different concentrations of the extract were either added at the start of the incubation time (24 h group) or 2 h prior to the end of the incubation time (2 h group). Afterwards cells were washed with Dulbecco’s phosphate-buffered saline (DPBS) and incubated for 2 h with fresh media containing 100 mM t-BOOH. Cells were washed again with DPBS and incubated for 2 h with 1 mM MTT solution diluted in RPMI medium without any additives. Thereafter the medium was removed and 100 ml 2-Propanol was added to each well and the plate was wobbled for 1 h at 450 rpm at RT. Absorption was measured 3 times with a Thermo Multiskan SPECTRUM plate reader at 570 nm.

2.8. Quantification of ROS The measurement of reactive oxygen species in SH-SY5Y cells was based on the 20 ,70 -dichlorodihydrofluorescein diacetate probe (H2DCFH-DA). Cells were seeded into the well of a 96-well microplate (4  104 cells/well) and incubated for 24 h. CRE was dissolved in RPMI 1640 medium and sterilely filtrated. Different concentrations of the extract were either added at the start of the incubation time (24 h group) or 2 h prior the end of the incubation time (2 h group). Afterwards cells were washed with DPBS and loaded with 20 mM H2DCFH-DA in the dark. Afterwards cells were washed two times with DPBS and medium containing 100 mM t-BOOH was added. Immediately after the addition of t-BOOH the fluorescence was recorded with a multi-detection plate reader (Mithras LB 940; Berthold) with an excitation wavelength of 485 nm and an emission wavelength of 528 nm. Changes in the fluorescence signal were recorded every 5 min for 120 min.

609

2.9. Quantification of apoptotic nuclei and mitochondrial membrane potential 3  105 Cells were seeded on gelatine (1% in DPBS) coated 24 mm round coverslips and incubated for 24 h in a humid atmosphere of 5% CO2 and 95% air at 37 1C. Extracts were either added at the start of the incubation time (24 h group) or 2 h prior the end (2 h group). Afterwards cells were washed with DPBS and incubated again for 2 h with fresh media containing 100 mM t-BOOH. Subsequently the cells were stained for 1 h with 25 nM Mitotracker Red CMX Ros, fixed for 20 min with 4% paraformaldehyde (PFA) and finally stained with 4 mM Hoechst 33342 for 10 min. Fluorescent images were obtained with a Leica microscope and an Axiovision camera. For Mitotracker CMX ROS a bandpass filter was used for excitation with a wavelength of 546 712 nm and a 590 nm longpass-emission-filter. Hoechst 33342 images were captured with an excitation wavelength of 365 nm and a 445750 nm emission bandpass filter. First the Mitotracker CMX ROS – then Hoechst 33342 – and finally the bright field image were recorded. All microscope and camera settings were kept constant during the entire experiment. After each image sequence the coverslip was moved at least 2.5 times the length of the visual field. For the quantification of the mitochondrial membrane potential, the intensity sum of the Mitotracker Red CMX Ros fluorescence was measured with image j (National Institutes of Health, http://rsbweb.nih.gov/ij/index) for each completely visible cell. Apoptosis was determined by scoring the Hoechst 33342 stained nuclei whereas uniformly stained un-deformed nuclei were considered as healthy, viable cells, while fragmented, deformed nuclei were counted as apoptotic nuclei. The experiment was repeated 4 times and at least 1600 cells were measured for each group with blinding of the investigator. For blinding, each image sequence became a randomized number and after data analysis those numbers were allocated to the specific group. 2.10. RNA isolation Cells were seeded in T-75 flasks (7.5  106 cells/flask) and incubated for 24 h without treatment (control) or with 100 mg/ml CRE (24 h group). Afterwards cells were harvested and the cell pellet was dissolved with 500 ml trizol in 2 ml tubes. 50 ml chloroform was added and the tube was shaken for 3 min followed by centrifugation for 10 min at 4 1C with 12,000g. The upper aqueous phase was transferred to a fresh 2 ml tube and 400 ml 96% isopropanol was added. After 10 min incubation at room temperature the mixture was centrifuged again for 10 min. at 4 1C with 12,000g. The supernatant was removed and the pellet was dissolved with 500 ml 70% ethanol, followed by another centrifugation step at 4 1C with 8000g for 5 min. Again the supernatant was discarded and the pellet was dried by air. The following steps were performed with the RNeasy Mini Kit (Quiagen; Hilden; Germany) according to the instructions of manufacturer. RNA concentration was adjusted to 100 ng/ml with RNase free water and the mixture was stored at  80 1C until further use. 2.11. Microarray-analysis The concentration and quality of the RNA were measured using a NanoDrop ND-1000 system (NanoDrop Technology). For genome-wide transcriptome analysis on Human Genome U219 microarrays (Affymetrix, Santa Clara, USA) cDNA synthesis, labeling and hybridization were carried out according to the 30 IVT Express Kit and the Hybridization, Wash and Stain Kit (Affymetrix) using 100 ng total RNA. The arrays were scanned on the Gene Atlas Platform using the Gene Atlas Instrument Control software (ver. 1.0.6.267). Signal processing was done using RMA background

610

T. Friedemann et al. / Journal of Ethnopharmacology 155 (2014) 607–615

correction and quantile normalization. For data analysis the probeset annotation (HGU219_Hs_ENSG_16.0.0) from the Brainarray database (http://brainarray.mbni.med.umich.edu/brainarray/Data base/ CustomCDF/genomic_curated_CDF.asp) was used. To identify significantly regulated genes, an unequal variance T-test with succeeding false discovery rate (FDR) correction was performed and the cut-off criteria defined as FDRo0.25 and a signal-log-ratio (SLR) 4 7 0.7. 2.12. RT-PCR Reverse transcription from RNA to cDNA was performed with high capacity RNA-to-cDNA Kit (Applied Biosystems) according to the manufacturers instructions. Diluted cDNA was semi-quantitatively analyzed using real-time PCR with the LightCycler 480 SYBR Green 1 Master (Roche) and Light Cycler 480 (Roche). GAPDH was used as an internal control. Primers were obtained from Eurofins MWG Operon (Ebersberg, Germany). The following human specific primers were used: TXNIP 50 -GATCACCGATTGGAGAGCCC-30 and 50 -TGCAGGGATCCACCTCAGTA-30 ; GAPDH 50 -GCATCTTCTTTTGCGTCGCC-30 and 50 -CCCAATACGACCAAATCCG TTG-30 . Each cycle of the RT-PCR included 10 s denaturation at 95 1C, 10 s primer annealing at 60 1C and 10 s synthesis at 72 1C. RT-PCR products were verified afterwards by a melting curve analysis (heating from 65 1C to 97 1C with 2.2 1C/s). The efficiency and cycle threshold was determined with LinRegPCR (Version 2012.3; Heart Failure Research Center Academic Medical Centre, Amsterdam, The Netherlands) and the relative expression ratio was calculated with the following equation (Schefe et al., 2006): rER ¼

Fig. 2 shows the CRE, NAC and trolox IC50 values of 224.2 713.0 mg/ml, 5.1 70.3 mg/ml and 5.6 7 0.6 mg/ml respectively. Radical scavenging activity of NAC and Trolox were significant superior to CRE (Po 0.01). The ORAC-assay was used to determine the peroxyl radical scavenging activity of the CRE. The results showed that 1 g of CRE has an antioxidant activity equal to 2.7 70.2 mM Trolox. 3.3. Effect of CRE on cell viability The cytotoxicity of the CRE was determined by treating the cells with different concentrations of the extract for 2 h or 24 h, followed by the MTT assay. Fig. 3 shows that a 2 h treatment of SH-SY5Y cells with 0–400 mg/ml CRE has only a small inhibitory effect on the cell viability. However, a 24 h treatment of the cells with CRE in different concentrations showed that 15 73.9% and 79.37 6.1% of the cells die at concentrations of 200 mg/ml and 400 mg/ml, respectively (Fig. 3; Po 0.05; Po 0.01; vs. medium control). Concentrations of 100 mg/ml and below showed no significant cytotoxic effect compared to the control.

Rnorm ðSOIÞ ð1 þ EðGOIÞÞ  ΔC T ðGOIÞ ¼ Rnorm ðREFÞ ð1 þEðHKGÞÞ  ΔC T ðHKGÞ

with

ΔC T ðGenÞ ¼ C T ðGen; SOIÞ  C T ðGen; REFÞ rER: relative expression ratio; Rnorm: fluorescence intensity; SOI: sample; GOI: target gene; REF: reference sample; HKG: housekeeping gene; CT: cycle threshold; E: mean efficiency. The results are from three independent experiments measured in triplicate. 2.13. Statistics The results are expressed as means 7SEM of n experiments. Statistical significance between sets of data was determined by ANOVA followed by the Bonferroni-Post-Hoc-Test. Differences were considered as statistically significant when P o0.05.

Fig. 1. High-performance liquid chromatogram of CRE and mixed standard compounds (MSC). (1) coptisine, (2) palmatine, (3) berberine. Chemical structures of the compounds were designed with ACD/ChemSketch (http://www.acdlabs.com/ resources/freeware/chemsketch/).

3. Results 3.1. HPLC-analysis The amount of the dried watery extract commensurate to 21.5% by weight of the dried herb was composed of 60.6 70.5 mg/g coptisine, 51.970.2 mg/g palmatine and 554.86 714.65 mg/g berberine. Fig. 1 shows a high-performance liquid chromatogram of the extract and of the mixed standard compounds. 3.2. Antioxidant activity of CRE The antioxidant activity of the CRE was determined by using the DPPH- and ORAC-assay. DPPH radical scavenging activity of the CRE was 3.6 70.2%; 5.2 7 0.1%; 7.4 70.1%; 13.8 70.7%; 26.2 70.1%; 48.3 70.3%; 81.0 7 4.2%; 95.1 72.0% at concentrations of 6.25; 12.5; 25; 50; 100; 200; 400; 800 mg/ml, respectively.

Fig. 2. DPPH-scavenging activity of trolox, n-acetylcystein (NAC) and CRE. Results represent the mean half-maximal inhibitory concentration (IC 50) 7 SEM of three independent experiments performed in triplicate. nn Indicates significant differences in comparison to CRE; P o 0.01.

T. Friedemann et al. / Journal of Ethnopharmacology 155 (2014) 607–615

Fig. 3. Effect of CRE on cell viability in SH-SY5Y neuroblastoma cells. Cells were pretreated for 24 h or 2 h with CRE (0–400 μg/ml) before t-BOOH (0 μM or 100 μM; 2 h) treatment. Thereafter cell viability was determined by 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide (MTT) staining. Results represent mean cell viability 7 SEM of six independent experiments performed in triplicate. n Po 0.05, nn Po 0.01 vs. control (0 μg/ml) and # Po 0.05, ## P o 0.01 2 h vs. 24 h of control— (0 μM t-BOOH) or treatment group (100 μM t-BOOH).

Fig. 4. Effect of CRE on ROS level in SH-SY5Y neuroblastoma cells. Cells were pretreated for 24 h or 2 h with CRE (100 μg/ml) before t-BOOH (100 μM; 2 h) treatment. ROS was determined by H2DCF-DA staining. Results are expressed as a percentage of control DCF fluorescence7 SEM of three independent experiments performed in triplicate.

The cytoprotective effect of CRE against t-BOOH damage was also evaluated with the MTT assay. Viability of cells exposed to 100 mM t-BOOH decreased to 54.2 71.7% (Po 0.01 vs. medium control) and increased significantly to 62.9 71.6% (Po 0.01 vs. t-BOOH control) in the 2 h pretreatment Group with 100 mg/ml CRE. The 24 h pretreatment Group showed that 100 mg/ml CRE even increased cell viability up to 71.9 72.3% (P o0.01 vs. t-BOOH control). Comparison of the 2 h and 24 h CRE groups revealed that the cytoprotective effect of CRE against t-BOOH damage was significantly better when the cells where pretreated for 24 h with the CRE (P o0.05). 3.4. Effect of CRE on intracellular reactive oxygen production Both the DPPH- and the ORAC-assays showed strong antioxidative activity of the CRE, therefore we investigated whether CRE can decrease the amount of ROS generated in cells exposed to 100 mM t-BOOH. As shown in Fig. 4 there was a significant increase of the fluorescence signal if the cells were exposed to t-BOOH

611

Fig. 5. Effect of CRE on t-BOOH-induced apoptosis. Results represent the percentage of apoptotic nuclei 7 SEM of 3 independent experiments. At least 1600 cells were analyzed in each group. n Po 0.05, nn P o0.01 in comparison to the control (no treatment); þ þ Po 0.01 vs. t-BOOH and ## P o0.01 vs. 2 h CRE.

Fig. 6. Effect of CRE on MMP. Cells were pretreated for 24 h or 2 h with CRE (100 μg/ml) before BOOH (100 μM; 2 h) treatment and subsequently stained with Mitotracker Red CMX ROS. MMP of cells without treatment (control) was set to 100%. Results are expressed as a percentage of control MMP 7 SEM of 3 independent experiments. At least 1600 cells were analyzed in each group. nn P o0.01 vs. control; þ þ Po 0.01 vs. t-BOOH and # Po 0.05 vs. 2 h CRE.

(P 40.01 vs. control), which indicates a significant ROS generation. However, neither the 2 h nor the 24 h pretreatment of cells exposed to t-BOOH with 100 mg/ml CRE demonstrated a significant reduction of the fluorescence signal. 3.5. Effect of CRE on apoptotic rate Results shown in Fig. 5 revealed that the apoptotic rate increased from 3.0 70.4% to 46.4 71.2% if the cells were treated with 100 mM t-BOOH (P o0.01 vs. medium control). Pretreatment with 100 mg/ml CRE for 2 h or 24 h significantly decreased the apoptotic rate to 37.37 1.7% and 28.1 71.4%, respectively (P 40.01 vs. t-BOOH control). Comparison of the 2 h and 24 h CRE pretreatment groups revealed that significantly fewer cells undergo apoptosis in the 24 h pretreatment group (P 40.05). 3.6. Effect of CRE on mitochondrial membrane potential Results showed that a 2 h treatment of SH-SY5Y cells with 100 mM t-BOOH results in a significant decrease of the fluorescence signal to 44.8 71.7% (P 40.01) compared to the medium

612

T. Friedemann et al. / Journal of Ethnopharmacology 155 (2014) 607–615

control which was set to 100% (Fig. 6). Pretreatment of the cells for 2 h or 24 h with 100 mg/ml CRE resulted in a significant increase of the MMP to 65.6 73.0% and 76.0 73.5% respectively (P o0.01 vs. tBOOH control). Comparison between the 2 h and 24 h pretreatment groups revealed that the loss of MMP is significantly more attenuated in the 24 h pretreatment group (P 40.05). 3.7. Transcriptional changes triggered by CRE Because the results of the cell viability, apoptosis and MMP measurements showed that 24 h of pretreatment of the cells was superior to 2 h of pretreatment, we investigated possible transcriptional changes triggered by CRE only in the 24 h group. Microarray datasets were evaluated with the probe-set annotation from the Brainarray database and we found two significant regulated genes (Table 1). Based on those results and an extensive literature research, which was looking for a link between the regulated genes and the neuroprotective effect of CRE, we decided to validate only TXNIP, one of the two significant regulated genes, by qRT-PCR. MTND1 was excluded because we could not find enough evidence in the literature which linked the up regulation of MTND1 with the present neuroprotective effect of CRE. However, the literature research revealed that down regulation of TXNIP protects cells from oxidative damage (Al-Gayyar et al., 2011; Gao et al., 2013). QRT-PCR results confirmed that TXNIP was significantly downwardly regulated with a signal log ratio (SLR) of  1.0 (P o0.01 vs. control; Fig. 7).

4. Discussion In the present study, we investigated the neuroprotective effect of CRE. The results showed that the watery extract protects SHSY5Y cells against t-BOOH-induced cytotoxicity, without significant reduction of the t-BOOH-induced ROS generation, by increasing cell viability, decreasing apoptosis and attenuation of the mitochondrial membrane depolarization. Additionally, we investigated transcriptional changes in response to CRE treatment and found out that TXNIP was significantly down regulated. The cytotoxicity of t-BOOH is based on the iron-dependent conversion into tert-butoxyl radicals, resulting in lipid peroxidation and intracellular ROS production (Nieminen et al., 1997; Amoroso et al., 1999; Martin et al., 2001; Annunziato et al., 2003). Based on the literature, it could be expected that CRE attenuates oxidative stress by free radical and peroxyl radical scavenging compounds in the CRE (Hwang et al., 2002; Jung et al., 2009; Luo et al., 2011; Han et al., 2012). Therefore, we investigated the ability of CRE to reduce the amount of free radicals and peroxyl radicals with the DPPH-assay (Prior et al., 2005; Phipps et al., 2007) and the ORAC-assay (Huang et al., 2002). Our results showed that CRE captures free and peroxyl radicals in a concentration-dependent manner, which at the first glance leads to the hypothesis that CRE might protect cells from oxidative damage by its radical scavenging capacity. Drahota et al. (2005) showed that oxidative stress generated by t-BOOH lead to a reduction of cell viability and mitochondrial function. Therefore, we investigated the cytoprotective and

mitochondrial-protective effects of CRE by using the MTT-assay and measurement of the MMP. Our results showed that both 2 h and 24 h of pretreatment with 100 mg/ml CRE significantly increased cell viability compared to cells without pretreatment. Furthermore, t-BOOH-induced reduction of MMP was significantly attenuated in the 2 h and 24 h groups. These results suggest that the neuroprotective effect of the CRE is partly achieved by the perpetuation of the mitochondrial activity. Furthermore, we showed that the protective effect of CRE on cell viability and MMP was significantly more pronounced in the 24 h group than in the 2 h group. However, this contradicts the initial hypothesis that CRE might exert its neuroprotective effect by its radicalscavenging capacity. While ROS is generated in untreated cells continuously, although in low concentration, the amount of antioxidative substances from CRE decreases in the CRE-treated cells over time. If the neuroprotective effect would be based on the radical-scavenging capacity of the CRE, cells treated with CRE shortly before t-BOOH exposition should be protected better against the oxidative damage than cells treated for 24 h with CRE. Since this does not apply to the present experiments, we hypothesized that the neuroprotective effect was independent from the anti-oxidative properties of CRE and might be caused by CRE treatment-induced transcriptional changes. This hypothesis is strengthened by the results Zhou et al. (2008) and Zhang et al. (2009) published previously. Zhou et al. (2008) reported that berberine, the main component of CRE, protects PC-12 cells against oxygen-glucose deprivation induced ROS generation via inhibiting NMDA-receptor and not by its direct antioxidative properties. Zhang et al. (2009) revealed that the total base from Coptidis chinensis Franch. (CTB) and berberine (Ber) protected rats from neuronal damage induced by repeated intragastrically exposure of aluminium. He hypothesized that CTB and Ber antagonized the aluminum induced oxidative stress by downregulation of MAO-B mRNA and protein expression as well as reduction of MAO-B activity. To test whether the neuroprotective effect was independent from the anti-oxidative properties of CRE we investigated the

Fig. 7. Relative gene expression ratio (rER) of TXNIP (a) and BCLAF (b). Results represent mean rER 7SEM of three independent experiments performed in triplicates. nn indicates Po 0.01 vs. control.

Table 1 List of significant regulated genes using alternative probe-set annotation from the Brainarray database. UID

Gene symbol

Description

P-value

FDR

Signal-Log-Ratio

ENSG00000117289_at ENSG00000198888_at

TXNIP MTND1

thioredoxin interacting protein [Source: HGNC Symbol; Acc: 16952] mitochondrially encoded NADH dehydrogenase 1 [Source: HGNC Symbol; Acc: 7455]

8.13E  06 2.77E 05

0.03 0.04

 0.93 0.85

T. Friedemann et al. / Journal of Ethnopharmacology 155 (2014) 607–615

613

Fig. 8. ROS induced apoptosis via ASK1-Trx-TXNIP—and TXNIP-NLRP3 inflammasom-signaling pathway. ASK 1-induced apoptosis is inhibited by direct interaction with Trx. Oxidative stress induces the release of Trx from ASK1. This process is enhanced by TXNIP, which can bind to reduced Trx and inhibits the attachment of Trx to ASK1. If the oxidative stress is rather high, the Trx-TXNIP complex dissociates and TXNIP interacts with NLRP3. This interaction enables the assembling of the NLRP3 inflamatosom, caspase activation and as a result the release of IL-1β. Upon binding of IL-1β to the IL-1R a plurality of signaling pathways are triggered. Those pathways include the activation of ASK1, which stimulates apoptosis mediated cell death. APAF1, apoptotic peptidase activating factor 1; ATP, adenosine-50 -triphosphate ASK1, apoptosis signal regulating kinase 1; Bax, BCL2-associated X protein; Bcl2, B-cell CLL/lymphoma 2; CytC, mitochondrial cytochrome c; IL-1β, interleukin 1 beta; IL 1R, interleukin 1 receptor; JNK, c-Jun N-terminal kinase; JNK dTF, JNK dependent transcription factor; NLRP3, NLR family pyrin domain containing 3; p53, tumor protein p53; Trx, thioredoxin; TXNIP, thioredoxin interacting protein.

anti-oxidative properties of CRE with the cell-based H2DCFH-DA assays. Consistent with our hypothesis we could not prove that 2 h or 24 h pretreatment of SH-SY5Y cells with CRE can inhibit ROS generated by t-BOOH. This finding may suggest that the amount of anti-oxidative compounds in the extract is too low to have a significant influence on the ROS generation induced by the rather high dose of t-BOOH. To further test our hypothesis we investigated whether CRE attenuates apoptosis independently from an antioxidative mechanism. Oxidative damage by ROS leads to lipid peroxidation in the cell membrane. Once phospholipids of the mitochondrial membrane are attacked by radicals it results in a reduction of MMP and, as a consequence, cytochrome c can be released to the cytoplasm. Detachment of cytochrome c from the mitochondrial membrane triggers caspase 3 activation, which in turn leads to apoptosis (Dai et al., 1999; Li et al., 2006). The results presented in this study demonstrated that 2 h and 24 h pretreatment of the SHSY5Y cells significantly attenuates t-BOOH-induced apoptosis.

Thereby we determined that 24 h of pretreatment was significantly more effective than 2 h of pretreatment. The result that the neuroprotective effect of 24 h of pretreatment was more pronounced than 2 h of pretreatment, measured by cell viability assay, MMP assay and apoptosis assay, and the fact that CRE did not significantly influence ROS production strengthens our hypothesis that the neuroprotective effect may be caused by transcriptional changes induced due to the CRE treatment. To substantiate this hypothesis we conducted microarray analyses. The evaluation of the microarray data with the annotations from the Brainarray database generated a list of two significantly regulated genes (MTND1, TXNIP). Since MTND1 is an important protein of the mitochondrial complex 1 and it was reported that patients with Parkinsons disease (PD) show a decreased activity of the mitochondrial complex 1 in the substantia nigra (Richter et al., 2002), the present upward regulation of MTND1 could be interesting for the treatment of PD. However, an intensive literature research could not reveal a clear connection between MTND1

614

T. Friedemann et al. / Journal of Ethnopharmacology 155 (2014) 607–615

upward regulation and the neuroprotective effect of CRE. Therefore we excluded MTND1 from the further investigation. On the contrary, it is worth mentioning that the down regulation of TXNIP could be clearly connected with the present neuroprotective effect of CRE. TXNIP is a 50-kDa protein and belongs to the α-arrestin protein family. It was first identified in yeast as an inhibitor of thioredoxin (Trx), which plays a major role in intracellular ROSscavenging and thiol-reduction (Nordberg and Arnér, 2001; Ma, 2010; Lu and Holmgren, 2012) (Fig. 8). It was reported that Trx can directly influence the apoptotic signaling pathway by binding and thereby inactivating the apoptosis signal-regulating kinase (Ask1, also called mitogen-activated protein kinase kinase kinase 5) (Saitoh et al., 1998). Dissociation of the Trx-Ask1 complex results in c-Jun N-terminal kinase and p53 Map kinase pathway activation, which leads at the end to apoptosis (Lu and Holmgren, 2012). Oxidative stress promotes the translocation of TXNIP from the cytosol to the mitochondria. Once TXNIP reached the mitochondria it competes with Ask1 for the binding of Trx2, and nearly the same will happen in the cytosol with Trx1. This leads to the dissociation of the Trx-Ask1 complex and results in activation of apoptotic pathways (Saitoh et al., 1998; Saxena et al., 2010; Lu and Holmgren, 2012) (Fig. 8). Down regulation of TXNIP will reduce the amount of activated Ask1 and this will lead to attenuation of apoptotic signaling in the cell (Al-Gayyar et al., 2011; Gao et al., 2013). This suggests that down regulation of TXNIP could protect SH-SY5Y cells from oxidative injury by strengthening Trx-mediated suppression of ASK1 activation and the subsequent pathways (Fig. 8). Further qRT-PCR analysis of the TXNIP expression confirmed the microarray results, which shows clearly that 24 h treatment of SH-SY5Y cells with CRE leads to a significant down regulation of TXNIP. It is worth mentioning that down regulation of TXNIP should also have an influence on the cellular ROS-level, because the inhibition of the Trx redox-cycle by binding TXNIP to reduced Trx can be attenuated by the down regulation of TXNIX and this could reduce cellular oxidative stress (Yoshihara et al., 2010). However, in this study we could not find a significant effect of CRE on ROS generated by t-BOOH which is consistent with the study of Gao et al. (2013). They found that activation of the AMPactivated protein kinase (AMPK) potently inhibits the expression of TXNIP, which leads to an attenuation of activated ASK1 as well as the ASK1-P58 signaling pathway and protects against oxidative stress-induced podocytes injury without any significant change at the intracellular ROS-level (Gao et al., 2013). Further studies are needed to clarify the influence of down regulated TXNIP in the neuroprotective effect of CRE.

5. Conclusion In conclusion we demonstrated that t-BOOH treatment resulted in ROS generation, reduced cell viability, decreased MMP and apoptosis in human neuroblastoma cells. All those effects, with the exception of ROS production, were significantly attenuated by CRE. Furthermore, the results revealed that downward regulation of TXNIP may be essential for the neuroprotective effect. Downward regulation of this gene can reduce the inhibition of Trx by TXNIP and attenuate TXNIP-mediated activation of ASK1 which could protect the cells from ASK1-triggered apoptosis. Our results suggest that the down regulation of TXNIP by CRE plays an important role for its neuroprotective effect. Due to its neuroprotective properties CRE might be a potential therapeutic agent for supporting disease prevention associated with oxidative stress like diabetic neuropathy and neurodegenerative diseases, for instance Alzheimer’s disease or Parkinson’s disease. However, further studies are needed to reveal the detailed involvement of TXNIP in the neuroprotective effect of CRE.

Acknowledgements This study was supported by the Innovationsstiftung Hamburg. The authors would like to thank PD Dr. M. Jendrach (Experimental Neurology, Department of Neurology, Goethe University Hospital, Frankfurt am Main, Germany) for the SH-SY5Y cells and Christine Knies (Institute of Anatomy and Experimental Morphology; University Medical Center Hamburg-Eppendorf, Hamburg, Germany) for her technological support. We also acknowledge Prof. Dr. C. Lohr (Division of Animal Physiology; University of Hamburg; Hamburg, Germany) for his support in form and content.

References Al-Gayyar, M.M., Abdelsaid, M.A., Matragoon, S., Pillai, B.A., El-Remessy, A.B., 2011. Thioredoxin interacting protein is a novel mediator of retinal inflammation and neurotoxicity. British Journal of Pharmacology 164, 170–180. Amoroso, S., Gioielli, A., Cataldi, M., Di Renzo, G., Annunziato, L., 1999. In the neuronal cell line SH-SY5Y, oxidative stress-induced free radical overproduction causes cell death without any participation of intracellular Ca2 þ increase. Biochimica et Biophysica Acta 1452, 151–160. Amoroso, S., D‘Alessio, A., Sirabella, R., Di Renzo, G., Annunziato, L., 2002. Ca(2 þ )independent caspase-3 but not Ca(2 þ )-dependent caspase-2 activation induced by oxidative stress leads to SH-SY5Y human neuroblastoma cell apoptosis. Journal of Neuroscience Research 68, 454–462. Annunziato, L., Amoroso, S., Pannaccione, A., Cataldi, M., Pignataro, G., D‘Alessio, A., 2003. Apoptosis induced in neuronal cells by oxidative stress: role played by caspases and intracellular calcium ions. Toxicology Letters 139, 125–133. Chuang, W.C., Young, D.S., Liu, L.K., Sheu, S.J., 1996. Liquid chromatographic– electrospray mass spectrometric analysis of Coptidis Rhizoma. Journal of Chromatography A 755, 19–26. Coleman, J.B., Gilfor, D., Farber, J.L., 1989. Dissociation of the accumulation of singlestrand breaks in DNA from killing of cultured hepatocytes by an oxidative stress. Molecular Pharmacology 36, 193–200. Dai, J., Weinberg, R.S., Waxman, S., Jing, Y., 1999. Malignant cells can be sensitized to undergo growth inhibition and apoptosis by arsenic trioxide through modulation of the glutathione redox system. Blood 93, 268–277. Department of Health of Hong Kong, 2008. Hong Kong Chinese Materia Medica Standards Vol 2, 278. Ding, P.L., Chen, L.Q., Lu, Y., Li, Y.G., 2012. Determination of protoberberine alkaloids in Rhizoma Coptidis by ERETIC 1H NMR method. Journal of Pharmaceutical and Biomedical Analysis 60, 44–50. Dong, S.K.J., 2000. Ming and Qing Chinese Masterpieces Series: Miraculous Recipe. (First Published 1449, Chinese Edition). Traditional Chinese Medicine press, Beijing, China. Drahota, Z., Krivakova, P., Cervinkova, Z., Kmonickova, E., Lotkova, H., Kucera, O., Houstek, J., 2005. Tert-butyl hydroperoxide selectively inhibits mitochondrial respiratory-chain enzymes in isolated rat hepatocytes. Physiological Research 54, 67–72. Finkel, T., Holbrook, N.J., 2000. Oxidants, oxidative stress and the biology of ageing. Nature 408, 239–247. Gao, K., Chi, Y., Sun, W., Takeda, M., Yao, J., 2013. AMPK attenuates adriamycininduced oxidative podocyte injury through thioredoxin-mediated suppression of ASK1-P38 signaling pathway. Molecular Pharmacology, Epub ahead of print, 10.1124/mol.113.089458. Gillespie, K.M., Chae, J.M., Ainsworth, E.A., 2007. Rapid measurement of total antioxidant capacity in plants. Nature Protocols 2, 867–870. Gong, L.L., Fang, L.H., Wang, L.H., Sun, J.L., Qin, H.L., Li, X.X., Wang, S.B., Du, G.H., 2012. Coptisine exert cardioprotective effect through anti-oxidative and inhibition of RhoA/Rho kinase pathway on isoproterenol-induced myocardial infarction in rats. Atherosclerosis 222, 50–58. Gorman, A.M., McGowan, A., O‘Neill, C., Cotter, T., 1996. Oxidative stress and apoptosis in neurodegeneration. Journal of the Neurological Sciences 139, 45–52. Gu, X., Sun, J., Li, S., Wu, X., Li, L., 2013. Oxidative stress induces DNA demethylation and histone acetylation in SH-SY5Y cells: potential epigenetic mechanisms in gene transcription in Aβ production. Neurobiology of Aging 34, 1069–1079. Guidarelli, A., Cattabeni, F., Cantoni, O., 1997. Alternative mechanisms for hydroperoxide-induced DNA single strand breakage. Free Radical Research 26, 537–547. Han, A.M., Heo, H., Kwon, Y.K., 2012. Berberine promotes axonal regeneration in injured nerves of the peripheral nervous system. Journal of Medicinal Food 15, 413–417. Hattangady, N.G., Rajadhyaksha, M.S., 2009. A brief review of in vitro models of diabetic neuropathy. International Journal of Diabetes in Developing Countries 29, 143–149. Huang, D., Ou, B., Hampsch-Woodill, M., Flanagan, J.A., Prior, R.L., 2002. Highthroughput assay of oxygen radical absorbance capacity (ORAC) using a multichannel liquid handling system coupled with a microplate fluorescence reader in 96-well format. Journal of Agricultural and Food Chemistry 50, 4437–4444.

T. Friedemann et al. / Journal of Ethnopharmacology 155 (2014) 607–615

Huang, K.C., 1999. The Pharmacology of Chinese Herbs. CRC Press Inc., Boca Raton, FL, pp. 381–382. Hwang, J.M., Wang, C.J., Chou, F.P., Tseng, T.H., Hsieh, Y.S., Lin, W.L., Chu, C.Y., 2002. Inhibitory effect of berberine on tert-butyl hydroperoxide-induced oxidative damage in rat liver. Archives of Toxicology 76, 664–670. Jung, H.A., Min, B.S., Yokozawa, T., Lee, J.H., Kim, Y.S., Choi, J.S., 2009. Anti-Alzheimer and antioxidant activities of Coptidis Rhizoma alkaloids. Biological and Pharmaceutical Bulletin 32, 1433–1438. Kowaltowski, A.J., Castilho, R.F., Vercesi, A.E., 2001. Mitochondrial permeability transition and oxidative stress. FEBS Letters 495, 12–15. Li, J.J., Tang, Q., Li, Y., Hu, B.R., Ming, Z.Y., Fu, Q., Qian, J.Q., Xiang, J.Z., 2006. Role of oxidative stress in the apoptosis of hepatocellular carcinoma induced by combination of arsenic trioxide and ascorbic acid. Acta Pharmacologica Sinica 27, 1078–1084. Li, P., Nijhawan, D., Budihardjo, I., Srinivasula, S.M., Ahmad, M., Alnemri, E.S., 1997. Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade. Cell 91 (479–489), 1997. Li, S., 1999. Bencao Gangmu—Compendium of Materia Medica. (First Published 1593). vol. 2. Foreign Language Press, Beijing, China, pp. 1364–1378. Liu, X., Kim, C.N., Yang, J., Jemmerson, R., Wang, X., 1996. Induction of apoptotic program in cell-free extracts: requirement for dATP and cytochrome c. Cell 86, 147–157. Lo, Y.C., Shih, Y.T., Tseng, Y.T., Hsu, H.T., 2012. Neuroprotective effects of San-HuangXie-Xin-Tang in the MPP þ/MPTP models of Parkinson’s disease in vitro and in vivo. Evidence-Based Complementary and Alternative Medicine 2012, 1–10. Lu, J., Holmgren, A., 2012. Thioredoxin system in cell death progression. Antioxidants & Redox Signaling 17, 1738–1747. Luo, T., Zhang, H., Zhang, W.W., Huang, J.T., Song, E.L., Chen, S.G., He, F., Xu, J., Wang, H.Q., 2011. Neuroprotective effect of Jatrorrhizine on hydrogen peroxideinduced cell injury and its potential mechanisms in PC12 cells. Neuroscience Letters 498, 227–231. Ma, Q., 2010. Transcriptional responses to oxidative stress: pathological and toxicological implications. Pharmacology & Therapeutics 125, 376–393. Marinova, E.K., Nikolova, D.B., Popova, D.N., Gallacher, G.B., Ivanovska, N.D., 2000. Suppression of experimental autoimmune tubulointerstitial nephritis in BALB/c mice by berberine. Immunopharmacology 48, 9–16. Martin, C., Martinez, R., Navarro, R., Ruiz-Sanz, J.I., Lacort, M., Ruiz-Larrea, M.B., 2001. Tert-butyl hydroperoxide-induced lipid signaling in hepatocytes: involvement of glutathione and free radicals. Biochemical Pharmacology 62, 705–712. Miura, N., Yamamoto, M., Ueki, T., Kitani, T., Fukuda, K., Komatsu, Y., 1997. Inhibition of thymocyte apoptosis by berberine. Biochemical Pharmacology 53, 1315–1322. Nieminen, A.L., Byrne, A.M., Herman, B., Lemasters, J.J., 1997. Mitochondrial permeability transition in hepatocytes induced by t-BuOOH: NAD(P)H and reactive oxygen species. American Journal of Physiology. 272, 1286–1294. Nieminen, A.L., Saylor, A.K., Tesfai, S.A., Herman, B., Lemasters, J.J., 1995. Contribution of the mitochondrial permeability transition to lethal injury after exposure of hepatocytes to t-butylhydroperoxide. Biochemical Journal 307, 99–106. Nordberg, J., Arnér, E.S., 2001. Reactive oxygen species, antioxidants, and the mammalian thioredoxin system. Free Radical Biology and Medicine 31, 1287–1312. Perfeito, R., Cunha-Oliveira, T., Rego, A.C., 2012. Revisiting oxidative stress and mitochondrial dysfunction in the pathogenesis of Parkinson diseaseresemblance to the effect of amphetamine drugs of abuse. Free Radical Biology and Medicine 53, 1791–1806. Perry, G., Cash, A.D., Smith, M.A., 2002. Alzheimer disease and oxidative stress. Journal of Biomedicine and Biotechnology 2, 120–123. Phipps, S.M., Sharaf, M.H., Butterweck, V., 2007. Assessing antioxidant activity in botanicals and other dietary supplements. Pharmacopeil Forum 33, 810–814.

615

Prior, R.L., Wu, X., Schaich, K., 2005. Standardized methods for the determination of antioxidant capacity and phenolics in foods and dietary supplements. Journal of Agricultural and Food Chemistry 53, 4290–4302. Richter, G., Sonnenschein, A., Grünewald, T., Reichmann, H., Janetzky, B., 2002. Novel mitochondrial DNA mutations in Parkinson’s disease. Journal of Neural Transmission 109, 721–729. Rush, G.F., Alberts, D., 1986. Tert-butyl hydroperoxide metabolism and stimulation of the pentose phosphate pathway in isolated rat hepatocytes. Toxicology and Applied Pharmacology 85, 324–331. Saitoh, M., Nishitoh, H., Fujii, M., Takeda, K., Tobiume, K., Sawada, Y., Kawabata, M., Miyazono, K., Ichijo, H., 1998. Mammalian thioredoxin is a direct inhibitor of apoptosis signal-regulating kinase (ASK) 1. EMBO Journal 17, 2596–2606. Saxena, G., Chen, J., Shalev, A., 2010. Intracellular shuttling and mitochondrial function of thioredoxin-interacting protein. Journal of Biological Chemistry 285, 3997–4005. Schefe, J.H., Lehmann, K.E., Buschmann, I.R., Unger, T., Funke-Kaiser, H., 2006. Quantitative real-time RT-PCR data analysis: current concepts and the novel “gene expression’s CT difference” formula. Journal of Molecular Medicine 84, 901–910. Seneviratne, C.J., Wong, R.W., Samaranayake, L.P., 2008. Potent anti-microbial activity of traditional Chinese medicine herbs against Candida species. Mycoses 51, 30–34. Sharma, O.P., Bhat, T.K., 2009. DPPH antioxidant assay revisited. Food Chemistry 113, 1202–1205. Smith, M.A., Perry, G., Richey, P.L., Sayrec, L.M., Anderson, V.E., 1996. Oxidative damage in Alzheimer’s. Nature 382, 120–121. Turkmen, N., Sari, F., Velioglu, Y.S., 2006. Effect of extraction solvents on concentration and antioxidant activity of black and black mate polyphenols determined by ferrous tartrate and Folin–Ciocalteu methods. Food Chemistry 99, 838–841. Vincent, A.M., Russell, J.W., Low, P., Feldman, E.L., 2004. Oxidative stress in the pathogenesis of diabetic neuropathy. Endocrine Reviews 25, 612–628. Wang, D., Liu, Z., Guo, M., Liu, S., 2004. Structural elucidation and identification of alkaloids in Rhizoma Coptidis by electrospray ionization tandem mass spectrometry. Journal of Mass Spectrometry 39, 1356–1365. Xie, H.R., Hu, L.S., Li, G.Y., 2010. SH-SY5Y human neuroblastoma cell line: in vitro cell model of dopaminergic neurons in Parkinson’s disease. Chinese Medical Journal 123, 1086–1092. Yoshihara, E., Chen, Z., Matsuo, Y., Masutani, H., Yodoi, J., 2010. Thiol redox transitions by thioredoxin and thioredoxin-binding protein-2 in cell signaling. Methods in Enzymology 474, 67–82. Zeng, H., Chen, Q., Zhao, B., 2004. Genistein ameliorates beta-amyloid peptide (25– 35)-induced hippocampal neuronal apoptosis. Free Radical Biology and Medicine 36, 180–188. Zhao, K., Luo, G., Giannelli, S., Szeto, H.H., 2005. Mitochondria-targeted peptide prevents mitochondrial depolarization and apoptosis induced by tert-butyl hydroperoxide in neuronal cell lines. Biochemical Pharmacology 70, 1796–1806. Zhang, J., Yang, J.Q., He, B.C., Zhou, Q.X., Yu, H.R., Tang, Y., Liu, B.Z., 2009. Berberine and total base from rhizoma coptis chinensis attenuate brain injury in an aluminum-induced rat model of neurodegenerative disease. Saudi Medical Journal 30, 760–766. Zhao, X., Nan, Y., Xiao, C., Zheng, J., Zheng, X., Wei, Y., Zhang, Y., 2010. Screening the bioactive compounds in aqueous extract of Coptidis rhizoma which specifically bind to rabbit lung tissues beta2-adrenoceptor using an affinity chromatographic selection method. Journal of Chromatography B 878, 2029–2034. Zhou, X.Q., Zeng, X.N., Kong, H., Sun, X.L., 2008. Neuroprotective effects of berberine on stroke models in vitro and in vivo. Neuroscience Letters 447, 31–36.