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Jujube-containing herbal decoctions induce neuronal differentiation and the expression of anti-oxidant enzymes in cultured PC12 cells
Author’s Accepted Manuscript Jujube-Containing Herbal Decoctions Induce Neuronal Differentiation and the Expression of Anti-oxidant Enzymes in Cultured PC12 cells Candy T.W. Lam, Amy G.W. Gong, Kelly Y.C. Lam, Laura M. Zhang, Jian-Ping Chen, Tina T.X. Dong, Huang-Quan Lin, Karl W.K. Tsim www.elsevier.com/locate/jep
PII: DOI: Reference:
S0378-8741(16)30278-1 http://dx.doi.org/10.1016/j.jep.2016.05.015 JEP10153
To appear in: Journal of Ethnopharmacology Received date: 1 February 2016 Revised date: 6 May 2016 Accepted date: 7 May 2016 Cite this article as: Candy T.W. Lam, Amy G.W. Gong, Kelly Y.C. Lam, Laura M. Zhang, Jian-Ping Chen, Tina T.X. Dong, Huang-Quan Lin and Karl W.K. Tsim, Jujube-Containing Herbal Decoctions Induce Neuronal Differentiation and the Expression of Anti-oxidant Enzymes in Cultured PC12 cells, Journal of Ethnopharmacology, http://dx.doi.org/10.1016/j.jep.2016.05.015 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Jujube-Containing Herbal Decoctions Induce Neuronal Differentiation and the Expression of Anti-oxidant Enzymes in Cultured PC12 cells
Candy T. W. Lama, Amy G. W. Gonga, Kelly Y. C. Lama, Laura M. Zhanga, Jian-Ping Chenb, Tina T. X. Donga, Huang-Quan Lina, Karl W. K. Tsima* a
Division of Life Science and Center for Chinese Medicine, The Hong Kong University of
Science and Technology, Hong Kong, China b
Pharmaceutical Department, Shenzhen Traditional Chinese Medicine Hospital, Guangzhou
University of Chinese Medicine, Shenzhen, China
*Corresponding Author: Prof. Karl W. K. Tsim, Division of Life Science and Center for Chinese Medicine, The Hong Kong University of Science and Technology, Clear Water Bay Road, Hong Kong, China Phone: +852- 2358 7332; fax: +852- 2358 1559; e-mail: [email protected].
Abstract ETHNOPHARMACOLOGICAL RELEVANCE: The fruit of Ziziphus jujuba (Mill.), known as Jujuba Fructus (JF) or jujube, is a well-known Traditional Chinese Medicine (TCM) for blood nourishment and sedation effect. Apart from prescribing as single herb alone, JF is very often being included in multi-herbal decoctions to prolong, enhance and harmonize pharmaceutical effects of decoctions while at the same time reducing toxicity. Here, we aimed to compare the protective and differentiating activities of three chemically standardized jujube-containing decoctions, including Guizhi Tang (GZT), Neibu Dangguijianzhong Tang (NDT) and Zao Tang (ZOT) in cultured PC12 cells. MATERIALS AND METHODS: The protein expressions of neurofilaments, including NF68, NF160 and NF200, under the herbal treatment were revealed by western blot. The determination of neurite outgrowth in 1
cultured PC12 cells upon the treatment of herbal extracts was performed by light microscope equipped with a phase-contrast condenser and SPOT imaging software. The protective effect against tBHP-induced cytotoxicity under the herbal treatment was measured by MTT assay. A luciferase reporter construct carrying four repeats of anti-oxidant response element (ARE) and a downstream luciferase reporter gene luc2P was transfected into PC12 cells to study the transcriptional activation of ARE. The mRNA expression of antioxidant enzymes under the herbal treatment was analyzed by quantitative real-time PCR. RESULTS: These jujube-containing decoctions processed similar neuro-protective and brain beneficial properties. The herbal treatment induced the protein expression of neurofilaments (NFs). Neurite outgrowth was observed under the herbal treatment. In parallel, the pre-treatment of herbal extracts protected PC 12 cells against oxidative stress-induced apoptosis in a dose-dependent manner. Moreover, the herbal treatments triggered the mRNA expressions of relevant anti-oxidation genes, i.e. glutamate-cysteine ligase catalytic subunit (GCLC), glutamate-cysteine ligase modulatory subunit (GCLM), glutathione S-transferase (GST) and NAD(P)H quinone oxidoreductase (NQO1) via the activation of anti-oxidant response element (ARE).
CONCLUSION: The results therefore demonstrated neuro-protective and differentiating properties of the three closely related decoctions, and which subsequently illustrated the enhancement function of jujube within a multi-herbal decoction.
Keywords:
Jujuba Fructus; differentiation
Decoction;
Anti-oxidation;
Neurofilament;
Neuronal
1. Introduction Neurogenesis is a complicated process involving the regeneration of neurons, neuronal differentiation, neuronal growth and survival (Barnea and Pravosudov, 2011; Glasper et al., 2
2012; Tirone et al., 2013). Unfortunately, once the neurogenesis process was impaired, it could cause loss of neurons and/ or inability of neuronal differentiation, resulting forgetfulness, poor memory, anxiety and neuronal cell death and leading to various neurodegenerative disorders like Parkinson’s, Alzheimer disease and depression (Parihar et al., 2013; Pristerà et al., 2013). Jujuba Fructus (JF), also known as jujube or Chinese date, is the fruit of Ziziphus jujuba (Mill.), and which is considered as one of five valuable fruits in China. JF serves as daily food, as well as being prescribed as a tonic medicine for blood nourishment and sedative effect. According to traditional Chinese medicine (TCM) theory, sedation is referring to calm the mind and to improve sleeping quality. Indeed, JF was reported to be the most frequently used herb in treating insomnia and in improving sleeping quality for over 2000 years (Yeung et al., 2012). In addition, flavonoids, saponins and polysaccharides, extracted from JF, was reported to possess sedative effect (Jiang et al., 2007a; Jiang et al., 2007b). In brain beneficial properties, the possessions of anti-oxidation and induction of neuronal differentiation are key parameters in explaining the neuro-protection effects. JF was reported to process anti-oxidation (Chen et al., 2013) and neuronal differentiation induction properties (Chen et al., 2014), which supported the neuro-protective function of jujube. However, the usage of jujube is not restricted as single herb only. It is very often being included in multi-herbal decoctions as an assistant, or a servant drug, to enhance the medicinal values, to facilitate the absorption, and/or to reduce the toxicity of individual herbs (Ung et al., 2007; Li et al., 2010). In the current studies, three jujube-containing decoctions were chosen for comparison. These herbal decoctions are commonly used today for similar clinical efficiency, i.e. Guizhi Tang (GZT), Neibu Dangguijianzhong Tang (NDT) and Zao Tang (ZOT). These jujube-containing decoctions were proposed to enhance hematopoietic function as well as brain beneficial effects (Lam et al., 2015). GZT was reported of having insomnia effect (Cui, 2006), and which was therapeutically used in treating cerebrovascular diseases (Hao et al., 2015). NDT was used for the treatment of insomnia and depression-associated with gastrointestinal neurosis (Guo, 2004). ZOT was reported with sedative and neuroprotective effect (Ceng et al., 2008a, Ceng et al., 2008b). GZT, composed of Cinnamomi Ramulus (CR; 3
Branch of Cinnamomum cassia Presl), Paeoniae Alba Radix (PAR; Root of Paeonia lactiflora Pall.), Glycyrrhizae Radix et Rhizoma Praeparata cum Melle (GRRPM; Root and Rhizome of Glycyrrhiza uralensis Fisch. or Glycyrrhiza inflata Bat.), Zingiberis Rhizoma Recens (ZRR; Rhizome of Zingiber officinale Rosc.) and JF, was prescribed in Shang Han Lun by Zhang Zhongjing in Han Dynasty (~200 AD). NDT, composed of Angelicae Sinensis Radix (ASR; root of Angelica sinensis (Oliv.) Diels.), Cinnamomi Cortex (CC; cortex of Cinnamomum cassia Presl.), PAR, GRRPM, ZRR and JF, was prescribed in Bei Ji Qian Jin Yao Fang by Sun Simiao in Tang Dynasty (652 AD). ZOT, composed of GRRPM, ZRR and JF, was prescribed in Formulae of Pharmacy Service for Great Peace and for Benefit of People by Official Bureau of Physicans (Taiyi Ju) in Sung Dynasty (1078~1085 AD). Here, we hypothesized that the neuro-protective function of jujube-containing decoctions might be very similar. In cultured PC12 cells, application of these herbal extracts could enhance: (i) anti-oxidation effect in preventing free radical-induced cell death; and (ii) induction of neurite outgrowth and expression of neurofilaments.
2. Material and methods 2.1 Herbal materials The fruits of Z. jujuba cv. Jinsixiaozao from Hebei of China were collected in 2012. The dried stem barks and dried young branches of C. cassia were collected from Guangxi China. The roots of Angelica sinensis were collected from Gansu. The roots and rhizomes of G. uralensis or G. inflata under the method for stir-baking with honey were collected from Inner Mongolia. The processed roots of P. lactiflora and the rhizomes of Z. officinale were purchased from herbal markets. The plant materials were authenticated by Dr. Tina Dong based on their morphological characteristics and deposited in the Center for Chinese Medicine, The Hong Kong University of Science and Technology.
2.2 Preparation of standardized herbal extracts Total herbs (25 g) were weighted according to the formulation and were boiled together under moderate heating in 20 volume of water for 1 hour, twice (Lam et al., 2015). The combined extracts were dried into powder under vacuum by lyophilizer at -80 oC. Before 4
biological assessments, the powder was re-dissolved with water to a concentration of 100 mg/mL, as stock solutions. The extracts were filtered through a 0.22 μm membrane filter before the cell treatment. The herbal extracts were chemically standardized as reported previously (Lam et al., 2015).
2.3 Cell culture Rat pheochromocytoma PC12 cells derived from adrenal gland were purchased from American Type Culture Collection (ATCC; Manassas, VA). The cells were cultured in Dulbecco’s modified Eagles medium (DMEM) supplemented with 100 IU/mL penicillin, 100 µg/mL streptomycin, 6% fetal bovine serum (FBS) and 6% horse serum (HS) in a humidified 7.5% CO2 incubator at 37 oC. For the neuronal differentiation assay, culture medium was depleted with supplement of 1% FBS, 1% HS, 100 U/mL penicillin and 100 μg/mL streptomycin for 3 hours before drug treatment. Culture reagents were from Invitrogen (Grand Island, NY). Cells were then treated with herbal extracts and/ or reagents for 48 hours.
2.4 Polyacrylamide gel electrophoresis (SDS-PAGE) To study the protein expression of neurofilaments under the treatment of various herbal extracts, PC12 cells were seeded into a 12-well plate in DMEM with 6% FBS, 6% HS, 100 IU/mL penicillin and 100 µg/mL streptomycin for 24 hours. Subsequently, the medium was depleted with low serum (1% FBS, 1% HS, 100 U/mL penicillin and 100 μg/mL streptomycin) for 3 hours. Herbal extracts were then applied to cultures for 48 hours. Cultures were collected in high salt lysis buffer (1M NaCl, 10mM HEPES, 1mM EDTA, 1mM EGTA, 0.5% Triton X-100 in pH 7.5). The lysate were shaken at 4 oC for 10 min, followed by centrifugation at 16,100 g for another 10 min at 4 oC. Total protein extracted from cultures was then quantified by protein assay using a kit from Bio-Rad Laboratories (Bio-Rad, Hercules, CA). Samples were then normalized according to the result obtained from the protein assay by high salt lysis buffer. Samples with equal amount of protein was added with equal volume of 2X direct lysis buffer (0.125 M HCl, 4% SDS, 20% glycerol, 2% 2-mercaptoethanol, 0.02% bromophenol blue and pH 6.8). The samples mixed with 2X direct lysis buffer were then 5
boiled at 95 oC for 5 min twice before electrophoresis. To study the expression of NF proteins, SDS-PAGE in a Mini-PROTEIN® III Cell with 6% acrylamide separating gel and 5% acrylamide stacking gel was employed. The gel was run in 1X SDS-PAGE running buffer (25 mM Tris, 0.192 M glycine, 0.1% SDS and pH 8.3) at 60 V (Chen et al., 2014) initially while the voltage increased to 80 V later once the bromophenol blue dye passed the stacking layer. Precision Plus Protein™ Pre-stained Dual Color Standards (Bio-Rad) with a range of 10 to 250 kDa was used.
2.5 Western blot analysis Following the SDS-PAGE electrophoresis separation, proteins were transferred to a NitroBind® nitrocellulose membrane (Bio-Rad) using a Mini Trans-Blot cell® at 40 V and 0.1 A overnight in 1X transfer buffer (24 mM Tris, 192 mM glycine, 15% ethanol, 0.1% SDS). Successful transfers of equal protein loading samples were confirmed by staining with Ponceau-S (0.3% ponceau S in 2% trichloroacetic acid solution). The nitrocellulose membrane was then blocked with 5% fat-free milk in Tris-Buffered Saline Tween 20 solution (TBS-T; 20mM Tris base, 137 mM NaCl, 0.1% Tween 20, pH 7.6) for 2 hours at room temperature. After that, the nitrocellulose membrane was incubated with diluted primary antibody in 2.5% fat free-milk in TBS-T overnight at 4 °C. Anti-neurofilament 200 (NF 200; Santa Cruz Biotechnology, Santa Cruz, CA), anti-NF 160 (Sigma, St. Louis, MO), anti-NF 68 (Sigma) and anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Abcam Ltd., Cambridge, UK) were employed as primary antibodies. Subsequently, the nitrocellulose membrane was rinsed with TBS-T for 1 hour and incubated with diluted horseradish peroxidase (HRP)-conjugated anti-mouse secondary antibody (Invitrogen) in 2.5% fat-free milk in TBS-T for another 2 hour at room temperature. The nitrocellulose membrane was washed with TBS-T again for 1 hour. The immune complexes were visualized using the enhanced chemiluminescence (ECL) method (GE Healthcare, Piscateway, NJ). The intensities of the bands of control and samples treated with herbal extracts running on gel were captured and determined utilized the ChemiDoc™ Touch Imaging System (Bio-Rad).
2.6 Neurite outgrowth assay 6
Cultured PC12 cells (5 x 104 cells/well) were seeded into a 35-mm plate in DMEM with 6% FBS, 6% HS, 100 IU/mL penicillin and 100 µg/mL streptomycin for 24 hours. Subsequently, the medium was depleted with low serum (1% FBS, 1% HS, 100 U/mL penicillin and 100 μg/mL streptomycin) for 3 hours. Herbal extracts were then applied to cultures for 48 hours. After treatment, cells were fixed with ice-cold 4% paraformaldehyde. A light microscope (Diagnostic Instruments, Sterling Heights, MI) equipped with a phase-contrast condenser, 10X objective lens, digital camera and SPOT imaging software was employed to reveal the morphological changes and the length of neurites. Approximately 100 cells were counted from at least 10 randomly chosen visual fields (Chen et al., 2014). The cells were marked as differentiated if one or more neurites were longer than the diameter of the cell body. The cells were classified into three categories according to their neurite length, including <15, 15-30, and >30µm.
2.7 Cell viability test Cultured PC12 cells were seed in 96-well plate with a density of 2 X 104 cells/ well for 24 hours. Cells were then pre-treated with different herbal extracts at different concentrations for 24 hours. After that, the cells were challenged with 150 µM tert-butyl hydroperoxide (tBHP) for 3 hours. Subsequently, cell viability was performed by the addition of MTT [3(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] in PBS at a final concentration of 0.5 mg/mL for 1 hour. The solution was then removed and the purple precipitate was then re-dissolved in DMSO. The cell viability was then determined by the optical density measurement using the spectrophotometer at 570 nm.
2.8 DNA construct and transfection Vector pGL4.37 [luc2P/ARE/Hygro] carrying four repeats of anti-oxidant response elements (ARE: 5’-TAG CTT GGA AAT GAC ATT GCT AAT GCT GCT GAG TCA ACT TT-3’) (Maiwulanjiang et al., 2014), and a luciferase reporter gene luc2P (Photinus pyralis) was named as pARE-Luc (Promega Corporation, Madison, WI). Cultured PC12 cells were transfected with pARE plasmid by Lipofectamine 3000 (Invitrogen) for 16 hours according to the manufacture’s guideline. The transfection efficiency was around 60% as determined by 7
another control plasmid carrying β-galactosidase with a downstream cytomegalovirus enhancer promoter sequence.
2.9 Luciferase assay Transfected PC12 cells were treated with various herbal extracts at different concentrations and/ or other reagents for 24 hours. The medium was removed and washed with PBS. The cells were lysed by lysis buffer (0.2% Triton X-100, 1 mM dithiothreitol, 100mM potassium phosphate buffer and pH 7.8) under shaking at 4 °C for 10 min. The samples were then centrifuged at 16,100 g for 10 min at 4 °C and the supernatant was collected for luciferase assay. Dual-Luciferase® Reporter Assay System (Applied Biosystems; Foster City, CA) was employed for analysis. The luciferase activity was normalized by the protein concentration of lysates.
2.10 Quantitative real time PCR PC12 cultures were treated with herbal extracts and/ or other reagent for 24 hours. Total RNA was isolated by RNAzol reagent from Molecular Research Center Inc. (Cincinnati, OH), followed by reverse transcription into cDNA according to the manufacturer’s instruction (Invitrogen). Real-time PCR was performed by using FastStart Universal SYBR Green Master (ROX) according to manufacturer’s instructions (Roche Diagnostics; Mannheim, Germany) (Maiwulanjiang et al., 2014). The primers for rat glutamate-cysteine catalytic subunit (GCLC) gene were 5’-CGT GGA CAC CCG ATG CAG TAT TCT G-3’ and 5’- CGT GGA CAC CCG ATG CAG TAT TCT G-3’ (261 bp; NM_010815.2); glutamate-cysteine ligase modulatory subunit (GCLM) gene were 5’ -CCT GCT GTG TGA TGC CAC CAG ATT TT-3’ and 5’- TCT GCT TTT CAC GAT GAC CGA GTA CC-3’ (197 bp; NM_017305.2); glutathione S-transferase (GST) gene were 5’- CCT GGG CAT CTG AAA CCT TTT GAG AC-3’ and 5’GCG AGC CAC ATA GGC AGA GAG C-3’ (180 bp; L29427); NAD(P)H quinone oxidoreductase (NQO1) gene were 5’- GAC CTT GCT TTC CAT CAC CAC CGG-3’ and 5’GTA GAG TGG TGA CTC CTC CCA GAC-3’ (241 bp; NM_017000.3). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal control and reference gene and the sequence was 5’- AAC GGA TTT GGC CGT ATT GG-3’ and 5’- CTT CCC GTT CAG 8
CTC TGG G-3’ (180 bp; L29427). SYBR green signal was detected by ABI 7500 fast real time PCR system (Applied Biosystems). Transcript levels were quantified by using ΔΔCt value method under Real time PCR 7500 software v2.0.6 in which the values of target genes were normalized first by GAPDH in same sample before comparison. The PCR products were analyzed by gel electrophoresis and melting curve analysis for confirmation of specific amplification.
2.11 Statistical analysis Individual data was expressed as mean ± SD. Statistical tests were performed with t-test (version 13.0, SPSS). Statistically significant changes were classified as significant (*) where p < 0.05, very significant (**) where p < 0.01, and highly significant (***) where p < 0.001.
3. Results Chemical standardization of herbal extract Three jujube-containing decoctions, i.e. GZT, NDT and ZOT, were chosen for analyses here. The composition of these herbal decoctions was shown in Table 1. Crude herbs were weighted separately according to different formulations to form a combined weight of 25 g. The mixed herbs was boiled in 20 volumes of water (v/w) for 1 hour and extracted twice. These herbal extracts were chemically standardized, as reported, previously (Lam et al., 2015). Twelve chemicals were subjected to analysis, i.e. JF-derived cAMP, cGMP and rutin; GRRPM-derived calycosin, formononetin, glycyrrhizic acid, and liquiritin; ZRR-derived 6-gingerol; CR/CC-derived cinnamic acid; PAR-derived paeoniflorin; ASR-derived ferulic acid and Z-ligustilide (Supplementary Fig. 1). HPLC fingerprint at absorbance of 210 nm was employed to standardize the decoctions (Supplementary Fig. 2). The abundant constituents found in these decoctions were liquiritin, glycyrrhizic acid and 6-gingerol. Besides, a LC-MS/MS method was employed to quantify the amounts of marker chemicals in decoctions, and the minimum requirement for 12 aforementioned chemicals within GZT, NDT and ZOT were developed (see Lam et al., 2015). The chemical standardization of herbal extract is a must as to ensure the repeatability of biochemical analyses.
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Jujube-containing decoction induces the neurite outgrowth In previous studies, PC12 cells were employed to study the promotion effect of jujube extract on neuronal differentiation (Chen et al., 2014). To investigate and compare the neuronal differentiation properties between jujube-containing decoctions and JF extract, the extract was applied onto cultured PC12 for 48 hours, and the protein expressions of three major neurofilament subunits, the markers for neuronal differentiation, including NF Light (NF68; ~68 kDa), NF Medium (NF160; ~160 kDa) and NF Heavy (NF200; ~200kDa), were revealed. Nerve growth factor (NGF), served as positive control, induced neurofilament expression in cultured PC12 cells (Fig. 1). Similar to NGF, the application of herbal extracts induced the expression of neurofilaments in a dose-dependent manner (Fig. 1). In general, jujube-containing herbal extracts demonstrated higher induction effect on neurofilament expression, as compared to JF extract alone. The maximal induction was achieved by ZOT at 2 to 3 mg/mL, giving ~150% increase of NF 68 (Fig. 1A) and NF 160 (Fig. 1B), and ~50% increase of NF 200 (Fig. 1C). The expression of GAPDH at ~38 kDa was used as an internal control for the experiment (Supplementary Fig. 3). The neuronal differentiation properties, triggered by the herbal extracts, were examined by morphological exploration under the microscope. Herbal extracts at 3 mg/mL were applied onto PC12 cultures for 48 hours. NGF at a concentration of 50 ng/mL, served as positive control, induced neuronal differentiation. As shown in Fig. 2A, all the herbal extracts could induce the neurite outgrowth of PC12 cells. The number of cells with an increase in neurite length under the treatment of ZOT was more significantly, as compared to other herbal extracts (Fig. 2B). The neurite induction was better in the treatment of ZOT.
Jujube-containing decoction shows anti-oxidative effect PC12 cell is a well-established model for studying the neuroprotection effect against oxidative stress insult. Tert-butyl hydroperoxide (tBHP), an oxidative stress inducer, was applied onto cultured PC 12 cells to induce neuronal cell death. The cell viability was determined by MTT assay. As shown in Fig. 3A, the number of living PC12 cells when exposed to tBHP was decreased in a dose-dependent manner. At a concentration of 150 µM tBHP treatment, about 20% of cells survived: this concentration therefore was used as a 10
routine inducer. Vitamin C, an anti-oxidant, at a concentration of 1 mM served as a positive control, giving about 50~60% cell viability (Fig. 3A insert). To reveal and compare the anti-oxidative effect of herbal extracts, the extracts were pre-treated to cultured PC12 cells for 24 hours before tBHP application. All the herbal extracts demonstrated protective effect against tBHP-induced cell death in a dose-dependent manner (Fig. 3B). Jujube-containing herbal extracts showed better protective effect, as compared to JF extract. The maximum protective effect was shown in ZOT at 2 to 3 mg/mL, giving ~70% cell viability. To investigate the underlying mechanism for anti-oxidative effect demonstrated by the herbal extracts, the nuclear factor E2-related factor 2/ anti-oxidant response element (Nrf2/ARE) signaling pathway was revealed (Nguyen et al., 2009). ARE is an enhancer sequence or cis-acting regulatory element located in the promoter regions of genes encoding for anti-oxidant protein and detoxifying enzymes. The transcriptional activation of Nrf2/ARE signaling pathway stimulate the expressions of anti-oxidant proteins and detoxifying enzymes: this signaling is a defense mechanism in protecting cells from oxidative stress challenge. To illustrated the protective effect demonstrated by herbal extracts, the transcriptional activity of ARE was revealed here. Cultured PC12 cells were transfected with the reporter carrying four repeats of ARE and a downstream luciferase reporter gene (pARE-Luc) (Fig. 4 upper panel). The authentication of pARE-Luc was confirmed by activation activity under treatment of tert-Butylhydroquinone (tBHQ) in a dose-dependent manner (Fig. 4 insert). The herbal extracts were then applied onto pARE-Luc-transfected PC12 cells for 24 hours, which induced pARE-Luc activity in a dose-dependent manner (Fig. 4). The maximum induction was demonstrated by ZOT treatment at 3 mg/mL giving ~25 folds of induction in p-ARE-Luc transcriptional activity. To further extend our study, the role of herbal extracts in stimulating the gene expressions coding for anti-oxidant proteins and detoxifying enzymes were studied. The gene expression of ARE-derived genes, including GCLC, GCLM, GST and NQO1, were evaluated by real-time qPCR method. The herbal extracts were applied to cultured PC12 cells for 24 hours. Three µM tBHQ served as positive control here. The maximum induction of ARE-derived genes expression was achieved by ZOT application, while JF extract remained as the lowest induction. Under 3 mg/mL of ZOT, the levels of GCLC and GCLM 11
were increased by around 150% and 75%, respectively (Fig. 5A and 5B). The gene expressions of GST and NQO1, induced by ZOT, were even higher, i.e. over 200% (Fig. 5C and 5D).
4. Discussion According to theory of TCM in China, herbal decoction normally comprises of four elements, including “Jun” (prime), “Chen” (minister), “Zuo” (assistant) and “Shi” (servant). The four elements act together to harmonize the therapeutic functions. It was believed that herbal decoctions possess higher pharmaceutical value, as compared to single herb (Gao et al., 2007; Gong et al., 2015). JF, serving as assistant/servant herb, improved the efficacy of single herb by facilitate the absorption and reduced the toxicity of individual herbs (Dong et al., 2006; Li et al., 2010). In previous studies, jujube-containing decoctions demonstrated an enhancement of hematopoietic function, as compared to JF extract (Lam et al., 2015). Here, we aimed to explore the enhancement effect of jujube-containing decoctions on neuro-protective function by revealing anti-oxidation effect and neurofilament expression. Three jujube decoctions in our studies were written from different years with similar pharmacological functions, and they contained common herbal compatibility of GRRPM, ZRR and JF. The consistent usage of this herbal compatibility of having three herbs in herbal decoctions suggested its important role in disease healing. More important, the simplest formula of ZOT was the best in our cell assays. Neuronal differentiation is a crucial developmental process of a neuron in which the cell exhibits morphological changes with neurite formation and neurite outgrowth to form synapses for cell-cell communication. In various age-related neurological disorders, e.g. Alzheimer’s disease, synaptic dysfunction and neuronal loss have been reported (Schaeffer et al., 2010). Indeed, neuronal differentiation could be marked by the expression of neurofilaments. There are three NF subunits in supporting the elongation of neurites (Lin and Szaro, 1995), and they are defined according to their molecular weight, i.e. 68 kDa, 160 kDa and 200 kDa, respectively (Liu et al., 2004). The expression of NF68 takes place in the early stage of neurite outgrowth. Subsequently, NF160 expresses shortly with the emergence of 12
neurite formation. NF 200 appears in the late stage for axonal radial growth and nervous system maturation (Laser-Azogui et al., 2015). Three jujube-containing herbal extracts showed stronger effect in stimulating neurofilament expression, as compared to JF extract. Among the three decoctions, ZOT exhibited a more robust effect, especially in promoting the NF160 protein expression. Thus, the involvement of herbal extracts in neuronal differentiation could be mainly at the early stage, and which might not fully support the entire stage of neuronal differentiation. Reactive oxygen species (ROS) damage neuronal cells by generation of oxidative stress as to affect the cell survival (Yuan et al., 2014). The formation of ROS peroxidizes lipids and destroys the structural components of neuronal cells, e.g. protein, enzyme and nucleic acid and eventually contributing to neuron apoptosis, ageing or other neurological diseases (Schieber and Chandel, 2014; Trompier et al., 2014). To avoid heavy casualties of neurons, our body develops a defending mechanism towards the attack of ROS. The Nrf2/ARE signaling pathway triggers the production of anti-oxidant proteins and detoxifying enzymes, e.g. GCLC, GCLM, GST and NQO1 in order to get rid of ROS (Johnson et al., 2008). The jujube-containing herbal decoctions induced the ARE-derived anti-oxidation genes, including GCLC, GCLM, GST and NQO1, which showed better effect as compared to JF extract. Among the three decoctions, ZOT exhibited more significant effect, especially in promoting NQO1 expression. By expectation, the Nrf2/ARE signaling pathway could be triggered by the herbal extracts here. Thus, our current results support the brain beneficial functions of the jujube-containing decoctions.
5. Conclusions The result suggested that the herbal decoctions induced the neurite outgrowth by stimulating the neurofilament expression. Furthermore, the herbal decoctions possessed anti-oxidation effect and stimulating the gene expressions of anti-oxidation defense genes. The neuro-protective functions of the jujube-containing herbal decoctions, in particular the herbal decoction of ZOT, were higher than that of JF extract, which supported the rational of having a mixture of herbs. Three decoctions, described here, might serve as potential natural alternative medicine and healthy food supplement for patients with neurological disorders 13
6. Acknowledgments Supported by Hong Kong Research Grants Council Theme-based Research Scheme (T13-607/12R), ITF (UIM/254), GRF (661110, 662911, 660411, 663012, 662713, M-HKUST604/13), TUYF12SC02, TUYF12SC03, TUYF15SC01, The Hong Kong Jockey Club Charities Trust (HKJCCT12SC01) and Foundation of The Awareness of Nature (TAON12SC01) to Karl Tsim. Candy Lam received a scholarship from The Awareness of Nature Ltd. 7. References Barnea, A. and Pravosudov, V. (2011) Birds as a model to study adult neurogenesis: bridging evolutionary, comparative and neuroethological approches. European Journal of Neuroscience, 34: 884–907. Ceng, X.F., Liu, S.L., Li, D.S. (2008) The study of ginger-jujube-licorice combined prescription in Shanghan lun. Guide of China Medicine, 6:295-296. Ceng, X.F., Liu, S.L., Li, D.S. (2008) The study of ginger-jujube-licorice combined prescription in Jin kui yaolue. Journal of Traditional Chinese Medicine, 23:1599-1600. Chen, J., Li, Z., Maiwulanjiang, M., Zhang, W.L., Zhan, J.Y., Lam, C.T., Zhu, K.Y., Yao, P., Choi, R.C., Lau, D.T., Dong, T.T., Tsim, K.W. (2013) Chemical and biological assessment of Ziziphus jujuba fruits from China: different geographical sources and developmental stages. Journal of Agricultural and Food Chemistry, 61:7315-7324. Chen, J., Maiwulanjiang, M., Lam, K.Y., Zhang, W.L., Zhan, J.Y., Lam, C.T., Xu, S.L., Zhu, K.Y., Yao, P., Lau, D.T., Dong, T.T., Tsim, K.W. (2014) A standardized extract of the fruit of Ziziphus jujuba (Jujube) induces neuronal differentiation of cultured PC12 cells: a signaling mediated by protein kinase A. Journal of Agricultural and Food Chemistry, 62:1890-1897. Cui, X.L. (2006). The usage of Gui Zhi Tang in treating insomnia. Clinical Journal of Traditional Chinese Medicine, 18:431. Dong, T.T., Zhao, K.J., Gao, Q.T., Ji, Z.N., Zhu, T.T., Li, J., Duan, R., Cheung, A.W., Tsim, K.W. (2006) Chemical and biological assessment of a Chinese herbal decoction containing Radix Astragali and Radix Angelicae Sinensis: Determination of drug ratio in having optimized properties. Journal of Agricultural and Food Chemistry, 54:2767-2774. Gao, Q.T., Li, J., Cheung, J.K., Duan, J., Ding, A., Cheung, A.W., Zhao, K.J., Li, W.Z., 14
Dong, T.T.X., Tsim, K.W.K. (2007): Verification of the formulation and efficacy of Danggui Buxue Tang (a decoction of Radix Astragali and Radix Angelicae Sinensis): an exemplifying systematic approach to revealing the complexity of Chinese herbal medicine formulae. Chinese Medicine Journal, 2:12-22. Glasper, E.R., Schoenfeld, T.J., Gould, E. (2012) Adult neurogenesis: optimizing hippocampal function to suit the environment. Behavioral Brain Research, 227:380-383. Gong, A.G., Li, N., Lau, K.M., Lee, P.S., Yan, L., Xu, M.L., Lam, C.T., Kong, A.Y., Lin, H.Q., Dong, T.T., Tsim, K.W. (2015) Calycosin orchestrates the functions of Danggui Buxue Tang, a Chinese herbal decoction composing of Astragali Radix and Angelica Sinensis Radix: An evaluation by using calycosin-knock out herbal extract. Journal of Ethnopharmacology, 168:150-157. Guo, Z.F. (2004) Traditional Chinese Medicine in treating gastrointestinal neurosis. Journal for Beneficial Readings Drug Information & Medical Advices, 10: 19. Hao, Q.W., Ma, C.J., Di, Y.Q., Li, H., Zeng, H.B., Wan, Y. (2015). Progress of cardiovascular and cerebrovascular treatment by classical prescription of Guizhi decoction. World Chinese Medicine, 10:131-134. Jiang, J.G., Huang, X.J., Chen, J. (2007a) Separation and purification of saponins from Semen Ziziphus jujuba and their sedative and hypnotic effects. Journal of Pharmacy and Pharmacology, 59:1175-1180. Jiang, J.G., Huang, X.J., Chen, J., Lin, Q.S. (2007b) Comparison of the sedative and hypnotic effects of flavonoids, saponins, and polysaccharides extracted from Semen Ziziphus jujube. Natural Product Research, 21:310-320. Johnson, J.A., Johnson, D.A., Kraft, A.D., Calkins, M.J., Jakel, R.J., Vargas, M.R., Chen, P.C. (2008) The Nrf2-ARE pathway: an indicator and modulator of oxidative stress in neurodegeneration. Annals of the New York Academy of Science, 1147: 61-69. Lam, C.T., Chan, P.H., Lee, P.S., Lau, K.M., Kong, A.Y., Gong, A.G., Xu, M.L., Lam, K.Y., Dong, T.T., Lin, H., Tsim, K.W. (2016) Chemical and biological assessment of Jujube (Ziziphus jujuba)-containing herbal decoctions: Induction of erythropoietin expression in cultures. J Chromatography B doi: 10.1016/j.jchromb.2015.09.021. Li, S., Zhang, B., Jiang, D., Wei, Y., Zhang, N. (2010) Herb network construction and co-module analysis for uncovering the combination rule of traditional Chinese herbal formulae. BMC Bioinformatics, 11(Suppl 11): S6. Lin, W., Szaro, B.G. (1995) Neurofilaments help maintain normal morphologies and support elongation of neurites in Xenopus laevis cultured embryonic spinal cord neurons. The Journal of Neuroscience, 15:8331-8344. 15
Liua Q., Xie, F., Siedlak, S.L., Nunomurac, A., Hondaa, K., Moreiraa, P.I., Zhua, X., Smitha, M.A., Perry, G. (2004) Biomedicine and diseases: review neurofilament proteins in neurodegenerative diseases. Cellular and Molecular Life Sciences, 61: 3057–3075. Nguyen, T., Nioi, P., Pickett, C.B. (2009) The Nrf2-antioxidant response element signaling pathway and its activation by oxidative stress. The Journal of Biological Chemistry, 284: 13291–13295. Parihar, V.K., Hattiangady, B., Shuai, B., Shetty, A.K. (2013) Mood and memory deficits in a model of Gulf War illness are linked with reduced neurogenesis, partial neuron loss, and mild inflammation in the hippocampus. Neuropsychopharmacology, 38:2348-2362. Pristerà, A., Saraulli, D., Farioli-Vecchioli, S., Strimpakos, G., Costanzi, M., di Certo, M.G., Cannas, S., Ciotti, M.T., Tirone, F., Mattei, E., Cestari, V., Canu, N. (2013) Impact of N-tau on adult hippocampal neurogenesis, anxiety, and memory. Neurobiology of Aging, 34:2551-2563. Laser-Azogui, A., Kornreich, M., Malka-Gibor, E., Beck, R. (2015) Neurofilament assembly and function during neuronal development. Current Opinion in Cell Biology, 32:92-101. Li, S., Zhang, B., Jiang, D., Wei, Y., Zhang, N. (2010) Herb network construction and co-module analysis for uncovering the combination rule of traditional Chinese herbal formulae. BMC Bioinformatics, 11(Suppl 11): S6. Schaeffer, E.L., da Silva, E.R., Novaes Bde, A., Skaf, H.D., Gattaz, W.F. (2010) Differential roles of phospholipases A2 in neuronal death and neurogenesis: Implications for Alzheimer disease. Progress in Neuro-Psychopharmacology & Biological Psychiatry, 34:1381-1389. Schieber, M., Chandel, N.S. (2014) ROS function in redox signaling and oxidative stress. Current Biology, 24:R453-462. Tirone, F., Farioli-Vecchioli, S., Micheli, L., Ceccarelli, M., and Leonardi, L. (2013) Genetic control of adult neurogenesis: interplay of differentiation, proliferation and survival modulates new neurons function, and memory circuits. Frontiers in Cellular Neuroscience, 7: 00059. Trompier, D., Vejux, A., Zarrouk, A., Gondcaille, C., Geillon, F., Nury, T., Savary, S., Lizard, G. (2014) Brain peroxisomes. Biochimie, 98:102-110. Ung, C.Y., Li, H., Cao, Z.W., Li, Y.X., Chen, Y.Z. (2007) Are herb-pairs of traditional Chinese medicine distinguishable from others? Pattern analysis and artificial intelligence classification study of traditionally defined herbal properties. Journal of 16
Ethnopharmacology, 111:371-377. Yeung, W.F., Chung, K.F., Poon, M.M., Ho, F.Y., Zhang, S.P., Zhang, Z.J., Ziea, E.T., Wong, V.T. (2012) Chinese herbal medicine for insomnia: a systematic review of randomized controlled trials. Sleep Medicine Reviews, 16:497-507. Yuan, T.F., Gu, S., Shan, C., Marchado, S., Arias-Carrión, O. (2014) Oxidative stress and adult neurogenesis. Stem Cell Reviews and Reports, 11:706-709. Figure Legend: Figure 1 Herbal extracts induce protein expression of neurofilaments in PC12 cells. Cultured PC12 cells (2 × 105 cells/mL) were treated with herbal extracts (GZT, NDT, ZOT and JF from 0.5-3 mg/mL) for 48 hours. The cell lysates were collected to determine the protein expression of neurofilaments including NF-L (~68 kDa), NF-M (~160 kDa) and NF-H (~200 kDa) using specific antibody. NGF (50 ng/mL) served as the positive control (Upper panel). GAPDH (~38 kDa) served as loading control (Supplementary Figure 3). The neurofilament proteins were quantified from the blots by a calibrated densitometry. Values are expressed as the percentage of increase to basal reading (untreated culture), mean ± SD, n = 4. Statistical comparison are made with JF extract; * p < 0.05; ** p < 0.01; *** p < 0.001.
Figure 2 Herbal extracts induce neurite outgrowth. Culture PC12 cells were treated with herbal extracts (GZT, NDT, ZOT and JF) at a concentration of 3 mg/mL for 48 hours. The cultures were fixed with ice-cold 4% paraformaldehyde and the neurite outgrowth was observed by the elongation of neurites under microscope. (A): Cells were reviewed under microscope. Bar = 50 µm. (B): To quantify the differentiation effect, the lengths of neurite were counted. Data are expressed as % of cells in 100 counted cells, mean ± SD, n = 4. NGF (50 ng/mL) served as the positive control. Statistical
comparison
are
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JF
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* p < 0.05;
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Figure 3 Herbal extracts protect PC12 cell against tBHP induced cytotoxicity. 17
(A): Cultured PC12 cells (2 × 105 cells/well) were exposed to tBHP at various concentrations (0-300 µM) for 3 hours. Cell viability was determined by MTT assay and expressed as % of control (cells without tBHP). The concentration for routine analysis was indicated at 150 mM. (B): Cultured PC12 cells (2 × 105 cells/well) were pre-treated with herbal extracts (GZT, NDT, ZOT and JF) at various concentrations (0-3 mg/mL) for 24 hours before the addition of tBHP (150 mM) for cytotoxicity test as in (A). Values are expressed as % of control (untreated culture). Statistical comparison are made with JF extract; * p < 0.05; ** p < 0.01; *** p < 0.001.
Figure 4 Herbal extracts stimulate pARE-Luc transcriptional activity. A luciferase-reporter containing four repeats of AREs and a downstream luciferase-reporter gene, named as pARE-Luc, was used as a study tool. Culture PC12 cells transfected with pARE-Luc were treated with tBHQ at various concentrations (0-3 µM) for 24 hours. The cell lysates were collected. Luciferase assay were performed to measure ARE transcriptional activity (upper panel). In addition, cultured PC12 cells transfected with pARE-Luc were treated with herbal extracts (GZT, NDT, ZOT and JF from 0.5–3.0 mg/mL) for 24 hours. tBHQ (3 µM) served as a positive control. The cell lysates were subjected to luciferase assay. Values are expressed as the fold of increase to basal reading (untreated culture) and as mean ± SD, where n = 4, each with triplicate samples. Statistical comparison are made with JF extract; * p < 0.05; ** p < 0.01; *** p < 0.001.
Figure 5 Herbal extracts promote the gene expression of ARE–derived proteins. Culture PC12 cells were treated with herbal extracts (GZT, NDT, ZOT and JF) at various concentrations (0-3 mg/mL) for 24 hours. Total RNAs were extracted from cultures, followed by reverse transcription into cDNAs. Real time qPCR was conducted to determine the mRNA expression coding for GCLC, GCLM, GST and NQO1. Three µM tBHQ served as a positive control, while GAPDH was an internal control. Values are expressed as % of increase to basal reading (untreated culture), and in mean ± SD, where n = 4, each with triplicate
18
samples. Statistical comparison are made with JF extract; * p < 0.05; ** p < 0.01; *** p < 0.001. Supplementary Figure 1 The structure of marker chemicals. The chemical structures of 12 marker chemicals, including Jujuba Fructus (JF)-derived cAMP, cGMP, rutin; Glycyrrhizae Radix et Rhizoma Praeparata cum Melle (GRRPM)-derived calycosin,
formononetin,
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Supplementary Figure 2 Typical HPLC fingerprint of herbal extracts at 210 nm. Ten mg/mL herbal extract was subjected to HPLC analysis. The chemical fingerprint was revealed by a DAD detector at 210 nm. Liquiritin at 56 min, glycyrrhizic acid at 78 min and 6-gingerol at 83 min were identified in the HPLC fingerprint. Representative chromatograms are shown, n = 5.
Supplementary Figure 3 The protein expressions of GAPDH under the treatment of herbal extracts. GAPDH was used as an internal control for the neuronal differentiation studies. The protein expression remained unchanged. The control was performed in line to the protein analysis as described in Fig.1. Table 1. Historical formulation of jujube-containing decoction
Herb JF GRRPM ZRR CR CC PAR ASR
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PII: DOI: Reference:
S0378-8741(16)30278-1 http://dx.doi.org/10.1016/j.jep.2016.05.015 JEP10153
To appear in: Journal of Ethnopharmacology Received date: 1 February 2016 Revised date: 6 May 2016 Accepted date: 7 May 2016 Cite this article as: Candy T.W. Lam, Amy G.W. Gong, Kelly Y.C. Lam, Laura M. Zhang, Jian-Ping Chen, Tina T.X. Dong, Huang-Quan Lin and Karl W.K. Tsim, Jujube-Containing Herbal Decoctions Induce Neuronal Differentiation and the Expression of Anti-oxidant Enzymes in Cultured PC12 cells, Journal of Ethnopharmacology, http://dx.doi.org/10.1016/j.jep.2016.05.015 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Jujube-Containing Herbal Decoctions Induce Neuronal Differentiation and the Expression of Anti-oxidant Enzymes in Cultured PC12 cells
Candy T. W. Lama, Amy G. W. Gonga, Kelly Y. C. Lama, Laura M. Zhanga, Jian-Ping Chenb, Tina T. X. Donga, Huang-Quan Lina, Karl W. K. Tsima* a
Division of Life Science and Center for Chinese Medicine, The Hong Kong University of
Science and Technology, Hong Kong, China b
Pharmaceutical Department, Shenzhen Traditional Chinese Medicine Hospital, Guangzhou
University of Chinese Medicine, Shenzhen, China
*Corresponding Author: Prof. Karl W. K. Tsim, Division of Life Science and Center for Chinese Medicine, The Hong Kong University of Science and Technology, Clear Water Bay Road, Hong Kong, China Phone: +852- 2358 7332; fax: +852- 2358 1559; e-mail: [email protected].
Abstract ETHNOPHARMACOLOGICAL RELEVANCE: The fruit of Ziziphus jujuba (Mill.), known as Jujuba Fructus (JF) or jujube, is a well-known Traditional Chinese Medicine (TCM) for blood nourishment and sedation effect. Apart from prescribing as single herb alone, JF is very often being included in multi-herbal decoctions to prolong, enhance and harmonize pharmaceutical effects of decoctions while at the same time reducing toxicity. Here, we aimed to compare the protective and differentiating activities of three chemically standardized jujube-containing decoctions, including Guizhi Tang (GZT), Neibu Dangguijianzhong Tang (NDT) and Zao Tang (ZOT) in cultured PC12 cells. MATERIALS AND METHODS: The protein expressions of neurofilaments, including NF68, NF160 and NF200, under the herbal treatment were revealed by western blot. The determination of neurite outgrowth in 1
cultured PC12 cells upon the treatment of herbal extracts was performed by light microscope equipped with a phase-contrast condenser and SPOT imaging software. The protective effect against tBHP-induced cytotoxicity under the herbal treatment was measured by MTT assay. A luciferase reporter construct carrying four repeats of anti-oxidant response element (ARE) and a downstream luciferase reporter gene luc2P was transfected into PC12 cells to study the transcriptional activation of ARE. The mRNA expression of antioxidant enzymes under the herbal treatment was analyzed by quantitative real-time PCR. RESULTS: These jujube-containing decoctions processed similar neuro-protective and brain beneficial properties. The herbal treatment induced the protein expression of neurofilaments (NFs). Neurite outgrowth was observed under the herbal treatment. In parallel, the pre-treatment of herbal extracts protected PC 12 cells against oxidative stress-induced apoptosis in a dose-dependent manner. Moreover, the herbal treatments triggered the mRNA expressions of relevant anti-oxidation genes, i.e. glutamate-cysteine ligase catalytic subunit (GCLC), glutamate-cysteine ligase modulatory subunit (GCLM), glutathione S-transferase (GST) and NAD(P)H quinone oxidoreductase (NQO1) via the activation of anti-oxidant response element (ARE).
CONCLUSION: The results therefore demonstrated neuro-protective and differentiating properties of the three closely related decoctions, and which subsequently illustrated the enhancement function of jujube within a multi-herbal decoction.
Keywords:
Jujuba Fructus; differentiation
Decoction;
Anti-oxidation;
Neurofilament;
Neuronal
1. Introduction Neurogenesis is a complicated process involving the regeneration of neurons, neuronal differentiation, neuronal growth and survival (Barnea and Pravosudov, 2011; Glasper et al., 2
2012; Tirone et al., 2013). Unfortunately, once the neurogenesis process was impaired, it could cause loss of neurons and/ or inability of neuronal differentiation, resulting forgetfulness, poor memory, anxiety and neuronal cell death and leading to various neurodegenerative disorders like Parkinson’s, Alzheimer disease and depression (Parihar et al., 2013; Pristerà et al., 2013). Jujuba Fructus (JF), also known as jujube or Chinese date, is the fruit of Ziziphus jujuba (Mill.), and which is considered as one of five valuable fruits in China. JF serves as daily food, as well as being prescribed as a tonic medicine for blood nourishment and sedative effect. According to traditional Chinese medicine (TCM) theory, sedation is referring to calm the mind and to improve sleeping quality. Indeed, JF was reported to be the most frequently used herb in treating insomnia and in improving sleeping quality for over 2000 years (Yeung et al., 2012). In addition, flavonoids, saponins and polysaccharides, extracted from JF, was reported to possess sedative effect (Jiang et al., 2007a; Jiang et al., 2007b). In brain beneficial properties, the possessions of anti-oxidation and induction of neuronal differentiation are key parameters in explaining the neuro-protection effects. JF was reported to process anti-oxidation (Chen et al., 2013) and neuronal differentiation induction properties (Chen et al., 2014), which supported the neuro-protective function of jujube. However, the usage of jujube is not restricted as single herb only. It is very often being included in multi-herbal decoctions as an assistant, or a servant drug, to enhance the medicinal values, to facilitate the absorption, and/or to reduce the toxicity of individual herbs (Ung et al., 2007; Li et al., 2010). In the current studies, three jujube-containing decoctions were chosen for comparison. These herbal decoctions are commonly used today for similar clinical efficiency, i.e. Guizhi Tang (GZT), Neibu Dangguijianzhong Tang (NDT) and Zao Tang (ZOT). These jujube-containing decoctions were proposed to enhance hematopoietic function as well as brain beneficial effects (Lam et al., 2015). GZT was reported of having insomnia effect (Cui, 2006), and which was therapeutically used in treating cerebrovascular diseases (Hao et al., 2015). NDT was used for the treatment of insomnia and depression-associated with gastrointestinal neurosis (Guo, 2004). ZOT was reported with sedative and neuroprotective effect (Ceng et al., 2008a, Ceng et al., 2008b). GZT, composed of Cinnamomi Ramulus (CR; 3
Branch of Cinnamomum cassia Presl), Paeoniae Alba Radix (PAR; Root of Paeonia lactiflora Pall.), Glycyrrhizae Radix et Rhizoma Praeparata cum Melle (GRRPM; Root and Rhizome of Glycyrrhiza uralensis Fisch. or Glycyrrhiza inflata Bat.), Zingiberis Rhizoma Recens (ZRR; Rhizome of Zingiber officinale Rosc.) and JF, was prescribed in Shang Han Lun by Zhang Zhongjing in Han Dynasty (~200 AD). NDT, composed of Angelicae Sinensis Radix (ASR; root of Angelica sinensis (Oliv.) Diels.), Cinnamomi Cortex (CC; cortex of Cinnamomum cassia Presl.), PAR, GRRPM, ZRR and JF, was prescribed in Bei Ji Qian Jin Yao Fang by Sun Simiao in Tang Dynasty (652 AD). ZOT, composed of GRRPM, ZRR and JF, was prescribed in Formulae of Pharmacy Service for Great Peace and for Benefit of People by Official Bureau of Physicans (Taiyi Ju) in Sung Dynasty (1078~1085 AD). Here, we hypothesized that the neuro-protective function of jujube-containing decoctions might be very similar. In cultured PC12 cells, application of these herbal extracts could enhance: (i) anti-oxidation effect in preventing free radical-induced cell death; and (ii) induction of neurite outgrowth and expression of neurofilaments.
2. Material and methods 2.1 Herbal materials The fruits of Z. jujuba cv. Jinsixiaozao from Hebei of China were collected in 2012. The dried stem barks and dried young branches of C. cassia were collected from Guangxi China. The roots of Angelica sinensis were collected from Gansu. The roots and rhizomes of G. uralensis or G. inflata under the method for stir-baking with honey were collected from Inner Mongolia. The processed roots of P. lactiflora and the rhizomes of Z. officinale were purchased from herbal markets. The plant materials were authenticated by Dr. Tina Dong based on their morphological characteristics and deposited in the Center for Chinese Medicine, The Hong Kong University of Science and Technology.
2.2 Preparation of standardized herbal extracts Total herbs (25 g) were weighted according to the formulation and were boiled together under moderate heating in 20 volume of water for 1 hour, twice (Lam et al., 2015). The combined extracts were dried into powder under vacuum by lyophilizer at -80 oC. Before 4
biological assessments, the powder was re-dissolved with water to a concentration of 100 mg/mL, as stock solutions. The extracts were filtered through a 0.22 μm membrane filter before the cell treatment. The herbal extracts were chemically standardized as reported previously (Lam et al., 2015).
2.3 Cell culture Rat pheochromocytoma PC12 cells derived from adrenal gland were purchased from American Type Culture Collection (ATCC; Manassas, VA). The cells were cultured in Dulbecco’s modified Eagles medium (DMEM) supplemented with 100 IU/mL penicillin, 100 µg/mL streptomycin, 6% fetal bovine serum (FBS) and 6% horse serum (HS) in a humidified 7.5% CO2 incubator at 37 oC. For the neuronal differentiation assay, culture medium was depleted with supplement of 1% FBS, 1% HS, 100 U/mL penicillin and 100 μg/mL streptomycin for 3 hours before drug treatment. Culture reagents were from Invitrogen (Grand Island, NY). Cells were then treated with herbal extracts and/ or reagents for 48 hours.
2.4 Polyacrylamide gel electrophoresis (SDS-PAGE) To study the protein expression of neurofilaments under the treatment of various herbal extracts, PC12 cells were seeded into a 12-well plate in DMEM with 6% FBS, 6% HS, 100 IU/mL penicillin and 100 µg/mL streptomycin for 24 hours. Subsequently, the medium was depleted with low serum (1% FBS, 1% HS, 100 U/mL penicillin and 100 μg/mL streptomycin) for 3 hours. Herbal extracts were then applied to cultures for 48 hours. Cultures were collected in high salt lysis buffer (1M NaCl, 10mM HEPES, 1mM EDTA, 1mM EGTA, 0.5% Triton X-100 in pH 7.5). The lysate were shaken at 4 oC for 10 min, followed by centrifugation at 16,100 g for another 10 min at 4 oC. Total protein extracted from cultures was then quantified by protein assay using a kit from Bio-Rad Laboratories (Bio-Rad, Hercules, CA). Samples were then normalized according to the result obtained from the protein assay by high salt lysis buffer. Samples with equal amount of protein was added with equal volume of 2X direct lysis buffer (0.125 M HCl, 4% SDS, 20% glycerol, 2% 2-mercaptoethanol, 0.02% bromophenol blue and pH 6.8). The samples mixed with 2X direct lysis buffer were then 5
boiled at 95 oC for 5 min twice before electrophoresis. To study the expression of NF proteins, SDS-PAGE in a Mini-PROTEIN® III Cell with 6% acrylamide separating gel and 5% acrylamide stacking gel was employed. The gel was run in 1X SDS-PAGE running buffer (25 mM Tris, 0.192 M glycine, 0.1% SDS and pH 8.3) at 60 V (Chen et al., 2014) initially while the voltage increased to 80 V later once the bromophenol blue dye passed the stacking layer. Precision Plus Protein™ Pre-stained Dual Color Standards (Bio-Rad) with a range of 10 to 250 kDa was used.
2.5 Western blot analysis Following the SDS-PAGE electrophoresis separation, proteins were transferred to a NitroBind® nitrocellulose membrane (Bio-Rad) using a Mini Trans-Blot cell® at 40 V and 0.1 A overnight in 1X transfer buffer (24 mM Tris, 192 mM glycine, 15% ethanol, 0.1% SDS). Successful transfers of equal protein loading samples were confirmed by staining with Ponceau-S (0.3% ponceau S in 2% trichloroacetic acid solution). The nitrocellulose membrane was then blocked with 5% fat-free milk in Tris-Buffered Saline Tween 20 solution (TBS-T; 20mM Tris base, 137 mM NaCl, 0.1% Tween 20, pH 7.6) for 2 hours at room temperature. After that, the nitrocellulose membrane was incubated with diluted primary antibody in 2.5% fat free-milk in TBS-T overnight at 4 °C. Anti-neurofilament 200 (NF 200; Santa Cruz Biotechnology, Santa Cruz, CA), anti-NF 160 (Sigma, St. Louis, MO), anti-NF 68 (Sigma) and anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Abcam Ltd., Cambridge, UK) were employed as primary antibodies. Subsequently, the nitrocellulose membrane was rinsed with TBS-T for 1 hour and incubated with diluted horseradish peroxidase (HRP)-conjugated anti-mouse secondary antibody (Invitrogen) in 2.5% fat-free milk in TBS-T for another 2 hour at room temperature. The nitrocellulose membrane was washed with TBS-T again for 1 hour. The immune complexes were visualized using the enhanced chemiluminescence (ECL) method (GE Healthcare, Piscateway, NJ). The intensities of the bands of control and samples treated with herbal extracts running on gel were captured and determined utilized the ChemiDoc™ Touch Imaging System (Bio-Rad).
2.6 Neurite outgrowth assay 6
Cultured PC12 cells (5 x 104 cells/well) were seeded into a 35-mm plate in DMEM with 6% FBS, 6% HS, 100 IU/mL penicillin and 100 µg/mL streptomycin for 24 hours. Subsequently, the medium was depleted with low serum (1% FBS, 1% HS, 100 U/mL penicillin and 100 μg/mL streptomycin) for 3 hours. Herbal extracts were then applied to cultures for 48 hours. After treatment, cells were fixed with ice-cold 4% paraformaldehyde. A light microscope (Diagnostic Instruments, Sterling Heights, MI) equipped with a phase-contrast condenser, 10X objective lens, digital camera and SPOT imaging software was employed to reveal the morphological changes and the length of neurites. Approximately 100 cells were counted from at least 10 randomly chosen visual fields (Chen et al., 2014). The cells were marked as differentiated if one or more neurites were longer than the diameter of the cell body. The cells were classified into three categories according to their neurite length, including <15, 15-30, and >30µm.
2.7 Cell viability test Cultured PC12 cells were seed in 96-well plate with a density of 2 X 104 cells/ well for 24 hours. Cells were then pre-treated with different herbal extracts at different concentrations for 24 hours. After that, the cells were challenged with 150 µM tert-butyl hydroperoxide (tBHP) for 3 hours. Subsequently, cell viability was performed by the addition of MTT [3(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] in PBS at a final concentration of 0.5 mg/mL for 1 hour. The solution was then removed and the purple precipitate was then re-dissolved in DMSO. The cell viability was then determined by the optical density measurement using the spectrophotometer at 570 nm.
2.8 DNA construct and transfection Vector pGL4.37 [luc2P/ARE/Hygro] carrying four repeats of anti-oxidant response elements (ARE: 5’-TAG CTT GGA AAT GAC ATT GCT AAT GCT GCT GAG TCA ACT TT-3’) (Maiwulanjiang et al., 2014), and a luciferase reporter gene luc2P (Photinus pyralis) was named as pARE-Luc (Promega Corporation, Madison, WI). Cultured PC12 cells were transfected with pARE plasmid by Lipofectamine 3000 (Invitrogen) for 16 hours according to the manufacture’s guideline. The transfection efficiency was around 60% as determined by 7
another control plasmid carrying β-galactosidase with a downstream cytomegalovirus enhancer promoter sequence.
2.9 Luciferase assay Transfected PC12 cells were treated with various herbal extracts at different concentrations and/ or other reagents for 24 hours. The medium was removed and washed with PBS. The cells were lysed by lysis buffer (0.2% Triton X-100, 1 mM dithiothreitol, 100mM potassium phosphate buffer and pH 7.8) under shaking at 4 °C for 10 min. The samples were then centrifuged at 16,100 g for 10 min at 4 °C and the supernatant was collected for luciferase assay. Dual-Luciferase® Reporter Assay System (Applied Biosystems; Foster City, CA) was employed for analysis. The luciferase activity was normalized by the protein concentration of lysates.
2.10 Quantitative real time PCR PC12 cultures were treated with herbal extracts and/ or other reagent for 24 hours. Total RNA was isolated by RNAzol reagent from Molecular Research Center Inc. (Cincinnati, OH), followed by reverse transcription into cDNA according to the manufacturer’s instruction (Invitrogen). Real-time PCR was performed by using FastStart Universal SYBR Green Master (ROX) according to manufacturer’s instructions (Roche Diagnostics; Mannheim, Germany) (Maiwulanjiang et al., 2014). The primers for rat glutamate-cysteine catalytic subunit (GCLC) gene were 5’-CGT GGA CAC CCG ATG CAG TAT TCT G-3’ and 5’- CGT GGA CAC CCG ATG CAG TAT TCT G-3’ (261 bp; NM_010815.2); glutamate-cysteine ligase modulatory subunit (GCLM) gene were 5’ -CCT GCT GTG TGA TGC CAC CAG ATT TT-3’ and 5’- TCT GCT TTT CAC GAT GAC CGA GTA CC-3’ (197 bp; NM_017305.2); glutathione S-transferase (GST) gene were 5’- CCT GGG CAT CTG AAA CCT TTT GAG AC-3’ and 5’GCG AGC CAC ATA GGC AGA GAG C-3’ (180 bp; L29427); NAD(P)H quinone oxidoreductase (NQO1) gene were 5’- GAC CTT GCT TTC CAT CAC CAC CGG-3’ and 5’GTA GAG TGG TGA CTC CTC CCA GAC-3’ (241 bp; NM_017000.3). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal control and reference gene and the sequence was 5’- AAC GGA TTT GGC CGT ATT GG-3’ and 5’- CTT CCC GTT CAG 8
CTC TGG G-3’ (180 bp; L29427). SYBR green signal was detected by ABI 7500 fast real time PCR system (Applied Biosystems). Transcript levels were quantified by using ΔΔCt value method under Real time PCR 7500 software v2.0.6 in which the values of target genes were normalized first by GAPDH in same sample before comparison. The PCR products were analyzed by gel electrophoresis and melting curve analysis for confirmation of specific amplification.
2.11 Statistical analysis Individual data was expressed as mean ± SD. Statistical tests were performed with t-test (version 13.0, SPSS). Statistically significant changes were classified as significant (*) where p < 0.05, very significant (**) where p < 0.01, and highly significant (***) where p < 0.001.
3. Results Chemical standardization of herbal extract Three jujube-containing decoctions, i.e. GZT, NDT and ZOT, were chosen for analyses here. The composition of these herbal decoctions was shown in Table 1. Crude herbs were weighted separately according to different formulations to form a combined weight of 25 g. The mixed herbs was boiled in 20 volumes of water (v/w) for 1 hour and extracted twice. These herbal extracts were chemically standardized, as reported, previously (Lam et al., 2015). Twelve chemicals were subjected to analysis, i.e. JF-derived cAMP, cGMP and rutin; GRRPM-derived calycosin, formononetin, glycyrrhizic acid, and liquiritin; ZRR-derived 6-gingerol; CR/CC-derived cinnamic acid; PAR-derived paeoniflorin; ASR-derived ferulic acid and Z-ligustilide (Supplementary Fig. 1). HPLC fingerprint at absorbance of 210 nm was employed to standardize the decoctions (Supplementary Fig. 2). The abundant constituents found in these decoctions were liquiritin, glycyrrhizic acid and 6-gingerol. Besides, a LC-MS/MS method was employed to quantify the amounts of marker chemicals in decoctions, and the minimum requirement for 12 aforementioned chemicals within GZT, NDT and ZOT were developed (see Lam et al., 2015). The chemical standardization of herbal extract is a must as to ensure the repeatability of biochemical analyses.
9
Jujube-containing decoction induces the neurite outgrowth In previous studies, PC12 cells were employed to study the promotion effect of jujube extract on neuronal differentiation (Chen et al., 2014). To investigate and compare the neuronal differentiation properties between jujube-containing decoctions and JF extract, the extract was applied onto cultured PC12 for 48 hours, and the protein expressions of three major neurofilament subunits, the markers for neuronal differentiation, including NF Light (NF68; ~68 kDa), NF Medium (NF160; ~160 kDa) and NF Heavy (NF200; ~200kDa), were revealed. Nerve growth factor (NGF), served as positive control, induced neurofilament expression in cultured PC12 cells (Fig. 1). Similar to NGF, the application of herbal extracts induced the expression of neurofilaments in a dose-dependent manner (Fig. 1). In general, jujube-containing herbal extracts demonstrated higher induction effect on neurofilament expression, as compared to JF extract alone. The maximal induction was achieved by ZOT at 2 to 3 mg/mL, giving ~150% increase of NF 68 (Fig. 1A) and NF 160 (Fig. 1B), and ~50% increase of NF 200 (Fig. 1C). The expression of GAPDH at ~38 kDa was used as an internal control for the experiment (Supplementary Fig. 3). The neuronal differentiation properties, triggered by the herbal extracts, were examined by morphological exploration under the microscope. Herbal extracts at 3 mg/mL were applied onto PC12 cultures for 48 hours. NGF at a concentration of 50 ng/mL, served as positive control, induced neuronal differentiation. As shown in Fig. 2A, all the herbal extracts could induce the neurite outgrowth of PC12 cells. The number of cells with an increase in neurite length under the treatment of ZOT was more significantly, as compared to other herbal extracts (Fig. 2B). The neurite induction was better in the treatment of ZOT.
Jujube-containing decoction shows anti-oxidative effect PC12 cell is a well-established model for studying the neuroprotection effect against oxidative stress insult. Tert-butyl hydroperoxide (tBHP), an oxidative stress inducer, was applied onto cultured PC 12 cells to induce neuronal cell death. The cell viability was determined by MTT assay. As shown in Fig. 3A, the number of living PC12 cells when exposed to tBHP was decreased in a dose-dependent manner. At a concentration of 150 µM tBHP treatment, about 20% of cells survived: this concentration therefore was used as a 10
routine inducer. Vitamin C, an anti-oxidant, at a concentration of 1 mM served as a positive control, giving about 50~60% cell viability (Fig. 3A insert). To reveal and compare the anti-oxidative effect of herbal extracts, the extracts were pre-treated to cultured PC12 cells for 24 hours before tBHP application. All the herbal extracts demonstrated protective effect against tBHP-induced cell death in a dose-dependent manner (Fig. 3B). Jujube-containing herbal extracts showed better protective effect, as compared to JF extract. The maximum protective effect was shown in ZOT at 2 to 3 mg/mL, giving ~70% cell viability. To investigate the underlying mechanism for anti-oxidative effect demonstrated by the herbal extracts, the nuclear factor E2-related factor 2/ anti-oxidant response element (Nrf2/ARE) signaling pathway was revealed (Nguyen et al., 2009). ARE is an enhancer sequence or cis-acting regulatory element located in the promoter regions of genes encoding for anti-oxidant protein and detoxifying enzymes. The transcriptional activation of Nrf2/ARE signaling pathway stimulate the expressions of anti-oxidant proteins and detoxifying enzymes: this signaling is a defense mechanism in protecting cells from oxidative stress challenge. To illustrated the protective effect demonstrated by herbal extracts, the transcriptional activity of ARE was revealed here. Cultured PC12 cells were transfected with the reporter carrying four repeats of ARE and a downstream luciferase reporter gene (pARE-Luc) (Fig. 4 upper panel). The authentication of pARE-Luc was confirmed by activation activity under treatment of tert-Butylhydroquinone (tBHQ) in a dose-dependent manner (Fig. 4 insert). The herbal extracts were then applied onto pARE-Luc-transfected PC12 cells for 24 hours, which induced pARE-Luc activity in a dose-dependent manner (Fig. 4). The maximum induction was demonstrated by ZOT treatment at 3 mg/mL giving ~25 folds of induction in p-ARE-Luc transcriptional activity. To further extend our study, the role of herbal extracts in stimulating the gene expressions coding for anti-oxidant proteins and detoxifying enzymes were studied. The gene expression of ARE-derived genes, including GCLC, GCLM, GST and NQO1, were evaluated by real-time qPCR method. The herbal extracts were applied to cultured PC12 cells for 24 hours. Three µM tBHQ served as positive control here. The maximum induction of ARE-derived genes expression was achieved by ZOT application, while JF extract remained as the lowest induction. Under 3 mg/mL of ZOT, the levels of GCLC and GCLM 11
were increased by around 150% and 75%, respectively (Fig. 5A and 5B). The gene expressions of GST and NQO1, induced by ZOT, were even higher, i.e. over 200% (Fig. 5C and 5D).
4. Discussion According to theory of TCM in China, herbal decoction normally comprises of four elements, including “Jun” (prime), “Chen” (minister), “Zuo” (assistant) and “Shi” (servant). The four elements act together to harmonize the therapeutic functions. It was believed that herbal decoctions possess higher pharmaceutical value, as compared to single herb (Gao et al., 2007; Gong et al., 2015). JF, serving as assistant/servant herb, improved the efficacy of single herb by facilitate the absorption and reduced the toxicity of individual herbs (Dong et al., 2006; Li et al., 2010). In previous studies, jujube-containing decoctions demonstrated an enhancement of hematopoietic function, as compared to JF extract (Lam et al., 2015). Here, we aimed to explore the enhancement effect of jujube-containing decoctions on neuro-protective function by revealing anti-oxidation effect and neurofilament expression. Three jujube decoctions in our studies were written from different years with similar pharmacological functions, and they contained common herbal compatibility of GRRPM, ZRR and JF. The consistent usage of this herbal compatibility of having three herbs in herbal decoctions suggested its important role in disease healing. More important, the simplest formula of ZOT was the best in our cell assays. Neuronal differentiation is a crucial developmental process of a neuron in which the cell exhibits morphological changes with neurite formation and neurite outgrowth to form synapses for cell-cell communication. In various age-related neurological disorders, e.g. Alzheimer’s disease, synaptic dysfunction and neuronal loss have been reported (Schaeffer et al., 2010). Indeed, neuronal differentiation could be marked by the expression of neurofilaments. There are three NF subunits in supporting the elongation of neurites (Lin and Szaro, 1995), and they are defined according to their molecular weight, i.e. 68 kDa, 160 kDa and 200 kDa, respectively (Liu et al., 2004). The expression of NF68 takes place in the early stage of neurite outgrowth. Subsequently, NF160 expresses shortly with the emergence of 12
neurite formation. NF 200 appears in the late stage for axonal radial growth and nervous system maturation (Laser-Azogui et al., 2015). Three jujube-containing herbal extracts showed stronger effect in stimulating neurofilament expression, as compared to JF extract. Among the three decoctions, ZOT exhibited a more robust effect, especially in promoting the NF160 protein expression. Thus, the involvement of herbal extracts in neuronal differentiation could be mainly at the early stage, and which might not fully support the entire stage of neuronal differentiation. Reactive oxygen species (ROS) damage neuronal cells by generation of oxidative stress as to affect the cell survival (Yuan et al., 2014). The formation of ROS peroxidizes lipids and destroys the structural components of neuronal cells, e.g. protein, enzyme and nucleic acid and eventually contributing to neuron apoptosis, ageing or other neurological diseases (Schieber and Chandel, 2014; Trompier et al., 2014). To avoid heavy casualties of neurons, our body develops a defending mechanism towards the attack of ROS. The Nrf2/ARE signaling pathway triggers the production of anti-oxidant proteins and detoxifying enzymes, e.g. GCLC, GCLM, GST and NQO1 in order to get rid of ROS (Johnson et al., 2008). The jujube-containing herbal decoctions induced the ARE-derived anti-oxidation genes, including GCLC, GCLM, GST and NQO1, which showed better effect as compared to JF extract. Among the three decoctions, ZOT exhibited more significant effect, especially in promoting NQO1 expression. By expectation, the Nrf2/ARE signaling pathway could be triggered by the herbal extracts here. Thus, our current results support the brain beneficial functions of the jujube-containing decoctions.
5. Conclusions The result suggested that the herbal decoctions induced the neurite outgrowth by stimulating the neurofilament expression. Furthermore, the herbal decoctions possessed anti-oxidation effect and stimulating the gene expressions of anti-oxidation defense genes. The neuro-protective functions of the jujube-containing herbal decoctions, in particular the herbal decoction of ZOT, were higher than that of JF extract, which supported the rational of having a mixture of herbs. Three decoctions, described here, might serve as potential natural alternative medicine and healthy food supplement for patients with neurological disorders 13
6. Acknowledgments Supported by Hong Kong Research Grants Council Theme-based Research Scheme (T13-607/12R), ITF (UIM/254), GRF (661110, 662911, 660411, 663012, 662713, M-HKUST604/13), TUYF12SC02, TUYF12SC03, TUYF15SC01, The Hong Kong Jockey Club Charities Trust (HKJCCT12SC01) and Foundation of The Awareness of Nature (TAON12SC01) to Karl Tsim. Candy Lam received a scholarship from The Awareness of Nature Ltd. 7. References Barnea, A. and Pravosudov, V. (2011) Birds as a model to study adult neurogenesis: bridging evolutionary, comparative and neuroethological approches. European Journal of Neuroscience, 34: 884–907. Ceng, X.F., Liu, S.L., Li, D.S. (2008) The study of ginger-jujube-licorice combined prescription in Shanghan lun. Guide of China Medicine, 6:295-296. Ceng, X.F., Liu, S.L., Li, D.S. (2008) The study of ginger-jujube-licorice combined prescription in Jin kui yaolue. Journal of Traditional Chinese Medicine, 23:1599-1600. Chen, J., Li, Z., Maiwulanjiang, M., Zhang, W.L., Zhan, J.Y., Lam, C.T., Zhu, K.Y., Yao, P., Choi, R.C., Lau, D.T., Dong, T.T., Tsim, K.W. (2013) Chemical and biological assessment of Ziziphus jujuba fruits from China: different geographical sources and developmental stages. Journal of Agricultural and Food Chemistry, 61:7315-7324. Chen, J., Maiwulanjiang, M., Lam, K.Y., Zhang, W.L., Zhan, J.Y., Lam, C.T., Xu, S.L., Zhu, K.Y., Yao, P., Lau, D.T., Dong, T.T., Tsim, K.W. (2014) A standardized extract of the fruit of Ziziphus jujuba (Jujube) induces neuronal differentiation of cultured PC12 cells: a signaling mediated by protein kinase A. Journal of Agricultural and Food Chemistry, 62:1890-1897. Cui, X.L. (2006). The usage of Gui Zhi Tang in treating insomnia. Clinical Journal of Traditional Chinese Medicine, 18:431. Dong, T.T., Zhao, K.J., Gao, Q.T., Ji, Z.N., Zhu, T.T., Li, J., Duan, R., Cheung, A.W., Tsim, K.W. (2006) Chemical and biological assessment of a Chinese herbal decoction containing Radix Astragali and Radix Angelicae Sinensis: Determination of drug ratio in having optimized properties. Journal of Agricultural and Food Chemistry, 54:2767-2774. Gao, Q.T., Li, J., Cheung, J.K., Duan, J., Ding, A., Cheung, A.W., Zhao, K.J., Li, W.Z., 14
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Liua Q., Xie, F., Siedlak, S.L., Nunomurac, A., Hondaa, K., Moreiraa, P.I., Zhua, X., Smitha, M.A., Perry, G. (2004) Biomedicine and diseases: review neurofilament proteins in neurodegenerative diseases. Cellular and Molecular Life Sciences, 61: 3057–3075. Nguyen, T., Nioi, P., Pickett, C.B. (2009) The Nrf2-antioxidant response element signaling pathway and its activation by oxidative stress. The Journal of Biological Chemistry, 284: 13291–13295. Parihar, V.K., Hattiangady, B., Shuai, B., Shetty, A.K. (2013) Mood and memory deficits in a model of Gulf War illness are linked with reduced neurogenesis, partial neuron loss, and mild inflammation in the hippocampus. Neuropsychopharmacology, 38:2348-2362. Pristerà, A., Saraulli, D., Farioli-Vecchioli, S., Strimpakos, G., Costanzi, M., di Certo, M.G., Cannas, S., Ciotti, M.T., Tirone, F., Mattei, E., Cestari, V., Canu, N. (2013) Impact of N-tau on adult hippocampal neurogenesis, anxiety, and memory. Neurobiology of Aging, 34:2551-2563. Laser-Azogui, A., Kornreich, M., Malka-Gibor, E., Beck, R. (2015) Neurofilament assembly and function during neuronal development. Current Opinion in Cell Biology, 32:92-101. Li, S., Zhang, B., Jiang, D., Wei, Y., Zhang, N. (2010) Herb network construction and co-module analysis for uncovering the combination rule of traditional Chinese herbal formulae. BMC Bioinformatics, 11(Suppl 11): S6. Schaeffer, E.L., da Silva, E.R., Novaes Bde, A., Skaf, H.D., Gattaz, W.F. (2010) Differential roles of phospholipases A2 in neuronal death and neurogenesis: Implications for Alzheimer disease. Progress in Neuro-Psychopharmacology & Biological Psychiatry, 34:1381-1389. Schieber, M., Chandel, N.S. (2014) ROS function in redox signaling and oxidative stress. Current Biology, 24:R453-462. Tirone, F., Farioli-Vecchioli, S., Micheli, L., Ceccarelli, M., and Leonardi, L. (2013) Genetic control of adult neurogenesis: interplay of differentiation, proliferation and survival modulates new neurons function, and memory circuits. Frontiers in Cellular Neuroscience, 7: 00059. Trompier, D., Vejux, A., Zarrouk, A., Gondcaille, C., Geillon, F., Nury, T., Savary, S., Lizard, G. (2014) Brain peroxisomes. Biochimie, 98:102-110. Ung, C.Y., Li, H., Cao, Z.W., Li, Y.X., Chen, Y.Z. (2007) Are herb-pairs of traditional Chinese medicine distinguishable from others? Pattern analysis and artificial intelligence classification study of traditionally defined herbal properties. Journal of 16
Ethnopharmacology, 111:371-377. Yeung, W.F., Chung, K.F., Poon, M.M., Ho, F.Y., Zhang, S.P., Zhang, Z.J., Ziea, E.T., Wong, V.T. (2012) Chinese herbal medicine for insomnia: a systematic review of randomized controlled trials. Sleep Medicine Reviews, 16:497-507. Yuan, T.F., Gu, S., Shan, C., Marchado, S., Arias-Carrión, O. (2014) Oxidative stress and adult neurogenesis. Stem Cell Reviews and Reports, 11:706-709. Figure Legend: Figure 1 Herbal extracts induce protein expression of neurofilaments in PC12 cells. Cultured PC12 cells (2 × 105 cells/mL) were treated with herbal extracts (GZT, NDT, ZOT and JF from 0.5-3 mg/mL) for 48 hours. The cell lysates were collected to determine the protein expression of neurofilaments including NF-L (~68 kDa), NF-M (~160 kDa) and NF-H (~200 kDa) using specific antibody. NGF (50 ng/mL) served as the positive control (Upper panel). GAPDH (~38 kDa) served as loading control (Supplementary Figure 3). The neurofilament proteins were quantified from the blots by a calibrated densitometry. Values are expressed as the percentage of increase to basal reading (untreated culture), mean ± SD, n = 4. Statistical comparison are made with JF extract; * p < 0.05; ** p < 0.01; *** p < 0.001.
Figure 2 Herbal extracts induce neurite outgrowth. Culture PC12 cells were treated with herbal extracts (GZT, NDT, ZOT and JF) at a concentration of 3 mg/mL for 48 hours. The cultures were fixed with ice-cold 4% paraformaldehyde and the neurite outgrowth was observed by the elongation of neurites under microscope. (A): Cells were reviewed under microscope. Bar = 50 µm. (B): To quantify the differentiation effect, the lengths of neurite were counted. Data are expressed as % of cells in 100 counted cells, mean ± SD, n = 4. NGF (50 ng/mL) served as the positive control. Statistical
comparison
are
made
with
JF
extract;
* p < 0.05;
** p < 0.01;
*** p < 0.001.
Figure 3 Herbal extracts protect PC12 cell against tBHP induced cytotoxicity. 17
(A): Cultured PC12 cells (2 × 105 cells/well) were exposed to tBHP at various concentrations (0-300 µM) for 3 hours. Cell viability was determined by MTT assay and expressed as % of control (cells without tBHP). The concentration for routine analysis was indicated at 150 mM. (B): Cultured PC12 cells (2 × 105 cells/well) were pre-treated with herbal extracts (GZT, NDT, ZOT and JF) at various concentrations (0-3 mg/mL) for 24 hours before the addition of tBHP (150 mM) for cytotoxicity test as in (A). Values are expressed as % of control (untreated culture). Statistical comparison are made with JF extract; * p < 0.05; ** p < 0.01; *** p < 0.001.
Figure 4 Herbal extracts stimulate pARE-Luc transcriptional activity. A luciferase-reporter containing four repeats of AREs and a downstream luciferase-reporter gene, named as pARE-Luc, was used as a study tool. Culture PC12 cells transfected with pARE-Luc were treated with tBHQ at various concentrations (0-3 µM) for 24 hours. The cell lysates were collected. Luciferase assay were performed to measure ARE transcriptional activity (upper panel). In addition, cultured PC12 cells transfected with pARE-Luc were treated with herbal extracts (GZT, NDT, ZOT and JF from 0.5–3.0 mg/mL) for 24 hours. tBHQ (3 µM) served as a positive control. The cell lysates were subjected to luciferase assay. Values are expressed as the fold of increase to basal reading (untreated culture) and as mean ± SD, where n = 4, each with triplicate samples. Statistical comparison are made with JF extract; * p < 0.05; ** p < 0.01; *** p < 0.001.
Figure 5 Herbal extracts promote the gene expression of ARE–derived proteins. Culture PC12 cells were treated with herbal extracts (GZT, NDT, ZOT and JF) at various concentrations (0-3 mg/mL) for 24 hours. Total RNAs were extracted from cultures, followed by reverse transcription into cDNAs. Real time qPCR was conducted to determine the mRNA expression coding for GCLC, GCLM, GST and NQO1. Three µM tBHQ served as a positive control, while GAPDH was an internal control. Values are expressed as % of increase to basal reading (untreated culture), and in mean ± SD, where n = 4, each with triplicate
18
samples. Statistical comparison are made with JF extract; * p < 0.05; ** p < 0.01; *** p < 0.001. Supplementary Figure 1 The structure of marker chemicals. The chemical structures of 12 marker chemicals, including Jujuba Fructus (JF)-derived cAMP, cGMP, rutin; Glycyrrhizae Radix et Rhizoma Praeparata cum Melle (GRRPM)-derived calycosin,
formononetin,
glycyrrhizic
acid,
liquiritin;
Zingiberis
Rhizoma
Recens
(ZRR)-derived 6-gingerol; Cinnamomi Ramulus/ Cinnamomi Cortex (CR/CC)-derived cinnamic acid; Paeoniae Alba Radix (PAR)-derived paeoniflorin; Angelicae Sinensis Radix (ASR)-derived ferulic acid, Z-ligustilide, were shown.
Supplementary Figure 2 Typical HPLC fingerprint of herbal extracts at 210 nm. Ten mg/mL herbal extract was subjected to HPLC analysis. The chemical fingerprint was revealed by a DAD detector at 210 nm. Liquiritin at 56 min, glycyrrhizic acid at 78 min and 6-gingerol at 83 min were identified in the HPLC fingerprint. Representative chromatograms are shown, n = 5.
Supplementary Figure 3 The protein expressions of GAPDH under the treatment of herbal extracts. GAPDH was used as an internal control for the neuronal differentiation studies. The protein expression remained unchanged. The control was performed in line to the protein analysis as described in Fig.1. Table 1. Historical formulation of jujube-containing decoction
Herb JF GRRPM ZRR CR CC PAR ASR
Decoction formulae GZTa NDTa ZOTa 3g 1.3g 2.8g 4g 2.2g 8.3g 6g 6.8g 13.9g 6g 0 0 0 3.4g 0 6g 6.8g 0 0 4.5g 0 19
a
The herbal composition was calculated according to the historical record in total of 25 g for herbal preparation. Abbreviations: Guizhi Tang (GZT), Neibu Dangguijianzhong Tang (NDT) and Zao Tang (ZOT). Jujuba Fructus (JF), Glycyrrhizae Radix et Rhizoma Praeparata cum Melle (GRRPM), Zingiberis Rhizoma Recens (ZRR), Cinnamomi Ramulus (CR), Cinnamomi Cortex (CC), Paeoniae Alba Radix (PAR), Angelicae Sinensis Radix (ASR).
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