Liuwei Dihuang (LWDH), a Traditional Chinese Medicinal Formula, Protects against β-Amyloid Toxicity in Transgenic Caenorhabditis elegans

Liuwei Dihuang (LWDH), a classic Chinese medicinal formula, has been used to improve or restore declined functions related to aging and geriatric diseases, such as impaired mobility, vision, hearing, cognition and memory. Here, we report on the effect and possible mechanisms of LWDH mediated protection of β-amyloid (Aβ) induced paralysis in Caenorhabditis elegans using ethanol extract (LWDH-EE) and water extract (LWDH-WE). Chemical profiling and quantitative analysis revealed the presence of different levels of bioactive components in these extracts. LWDH-WE was rich in polar components such as monosaccharide dimers and trimers, whereas LWDH-EE was enriched in terms of phenolic compounds such as gallic acid and paeonol. In vitro studies revealed higher DPPH radical scavenging activity for LWDH-EE as compared to that found for LWDH-WE. Neither LWDH-EE nor LWDH-WE were effective in inhibiting aggregation of Aβ in vitro. By contrast, LWDH-EE effectively delayed Aβ induced paralysis in the transgenic C. elegans (CL4176) model which expresses human Aβ1–42. Western blot revealed no treatment induced reduction in Aβ accumulation in CL4176 although a significant reduction was observed at an early stage with respect to β-amyloid deposition in C. elegans strain CL2006 which constitutively expresses human Aβ1–42. In addition, LWDH-EE reduced in vivo reactive oxygen species (ROS) in C. elegans (CL4176) that correlated with increased survival of LWDH-EE treated N2 worms under juglone-induced oxidative stress. Analysis with GFP reporter strain TJ375 revealed increased expression of hsp16.2::GFP after thermal stress whereas a minute induction was observed for sod3::GFP. Quantitative gene expression analysis revealed that LWDH-EE repressed the expression of amy1 in CL4176 while up-regulating hsp16.2 induced by elevating temperature. Taken together, these results suggest that LWDH extracts, particularly LWDH-EE, alleviated β-amyloid induced toxicity, in part, through up-regulation of heat shock protein, antioxidant activity and reduced ROS in C. elegans.


Introduction
Liuwei Dihuang (LWDH) is a classic Chinese medicinal formula that has been used for more than a thousand years in China.It is comprised of 6 Chinese herbs: Radix Rehmanniae Praeparata (prepared root of Rehmannia glutiosa), Fructus Corni (fruit of Cornus officinalis), Cortex Moutan (root bark of Paeonia suffruticosa), Rhizoma Dioscoreae (rhizome of Dioscorea opposita), Poria (scleorotia of Poria cocos), and Rhizoma Alismatis (rhizome of Alisma plantago-aquatica).LWDH is orally administered as a decoction or in the form of pills.According to the theory of traditional Chinese medicine (TCM), LWDH has the properties of tonifying the ''Yin'' of kidney, the fundamental system to support reproduction, development and performance over life time.LWDH has been used to improve or restore declined functions related to aging process and geriatric diseases, such as impaired mobility, vision, hearing, cognition and memory [1].
Recently, a number of studies have revealed the beneficial effects and some possible mechanisms of action of LWDH.LWDH was shown to affect the learning and memory function of senescence accelerated mice (SAM) primarily by altering the expression of a number of genes in hippocampus [2][3][4][5].Serum of LWDH-treated rats had positive effect on neuronal and synaptic functions in cultured rat hippocampal neurons [6].In addition, LWDH modulated the hypothalamus-pituitary-ovary axis in SAM model by changing the concentration of peptide neuro-mediators and estradiol receptor (ER-a) in pituitary and ovary and thus exerted the anti-aging effect [7].Also using SAM model, Feng and Zang [8] demonstrated that LWDH decreased the concentration of interleukins (IL-2 and IL-6) in hippocampus suggesting this as one possible mechanism through which LWDH may improve learning, performance, and memory.In scopolamine (SCOP), pchloroamphetamine (PCA), and cycloheximide (CXM)-induced amnesia rat models LWDH is believed to activate peripheral cholinergic neuronal system and modulate the central nervous system [9,10].More recently, in D-galactose-induced aging mouse and rat models, it was observed that LWDH treatment markedly improved the impaired learning and memory functions, possibly through restoring acetylcholine levels and cholineacetyltransferase activity, inhibiting acetylcholinesterase, and enhancing antioxidant activities in the brain [11][12][13].In aluminum-induced dementia rat model, LWDH treatment alleviated the memory impairment by concomitantly increasing plasma SOD activity and reducing MDA levels and protected the brain from damage due to lipid peroxidation [14].
Model organisms that mimic human responses offer tremendous opportunities for testing the potential role of natural products in neurodegenerative diseases.Caenorhabditis elegans (Nematoda: Rhabditidae) is an advantageous, cost effective animal model as it has a short lifespan, a very well characterized genome and can be cultured with ease in the laboratory.Interestingly there is a considerable homology between the C. elegans and human genomes [15] and as such C. elegans has been successfully used as a model system to study aging and age-associated neurodegenerative diseases [16][17][18].Transgenic C. elegans strains that express human b-amyloid (Ab) has been used to understand molecular mechanisms of Ab toxicity and also to screen for therapeutic agents aimed to ameliorate Ab toxicity [19][20][21].During the past decade, the utility of C. elegans has been eloquently used to study the health promoting effects of natural products used in TCM such as extracts from Ginkgo biloba, Cinnamomum cassia bark and Panax ginseng root [20,[22][23][24][25][26][27][28][29][30][31][32].Similarly, green tea component epigallocatechin gallate [33,34] and coffee extract [35], as well as other natural compounds like soy isoflavone [36], reserpine [37], and glaucarubinone [38] were shown to increase lifespan and stress resistance, and protect C. elegans against Ab toxicity.No study, to present date, has been done on the protective effect of LWDH against Ab in C. elegans model.Here we report our findings examining the effects of the major chemical constituents of water and ethanol extracts of LWDH (LWDH-WE and LWDH-EE) on antioxidant activity and Ab aggregation.Using a transgenic C. elegans model of AD, we tested for the protective effects of LWDH-WE and LWDH-EE against Ab toxicity and elucidated some of the mechanisms involved in delayed paralysis.
The LWDH extracts were prepared as follows: the dry unpolished pills (6.9 kg) were milled and extracted twice with 95% EtOH with refluxing (30 min each, total EtOH 45 L), the solvent was then removed under reduced pressure and dried at 70uC to obtain LWDH EtOH extract (LWDH-EE, 1.12 kg).The pill powder that was left over after EtOH extraction was extracted with boiling water (30 min each, total water 60 L), the water was then removed at reduced pressure and dried at 70uC to yield LWDH water extract (LWDH-WE, 2.24 kg).
Chemical profiling of LWDH extracts was done with NMR and HPLC-MS methods.For 1 H-NMR analysis, LWDH-EE was dissolved in methanol-d 4 , while LWDH-WE was dissolved in D 2 O. NMR spectra were acquired on a Bruker Avance III 600 MHz NMR spectrometer (Bruker Corporation, East Milton, ON) operating at 600.28 MHz 1 H observation frequency and a temperature of 2560.2uC.The signals were acquired, processed and analyzed using TopSpinH NMR data acquisition and processing Software (Bruker Biospin Ltd, East Milton, ON) integrated with the spectrometer.
For high performance liquid chromatography -mass spectroscopy (HPLC-MS) analysis, two types of columns were used.First, separation was conducted on an Agilent Zorbax SB-C18 RRHD (2.16150 mm, 1.8 mm) column using Agilent HPLC 1100 with diode array detector (DAD) and mass selective detector (MSD) systems.Solvent A was water with 0.1% formic acid and solvent B was acetonitrile with 0.1% formic acid.Gradient elution started with 2% of solvent B for 5 min, and increased to 30% solvent B in 55 min and then to 100% solvent B in 20 min, with a total run time of 80 min.Column temperature was 30uC, flow rate was 0.2 mL/min.Mass spectra were obtained on Agilent MSD under the following conditions: drying gas flow (L/min): 10; nebulizer pressure (psig): 30; drying gas temperature (uC): 350; capillary voltage (V): 4000 (positive) and 3500 (negative).Separately, a Phenomenex Luna hydrophilic interaction liquid chromatography (HILIC) column (25064.60mm, 5 mm) was used for analysis using Agilent HPLC 1290 with 1200 series DAD and evaporative laser scattering detector (ELSD) systems.Solvent A was acetonitrile/water/50 mM ammonium acetate (pH 3.2) (50/40/10) and solvent B was acetonitrile/50 mM ammonium acetate (pH 3.2) (90/10).The gradient scheme was 0% A/100% B to 100% A/0% B in 50 min.Column temperature was 30uC, and flow rate was 0.5 mL/min.To obtain MS information, the same HPLC condition was applied for Agilent 1100 HPLC with MSD system.Parameters of MSD were the same as described above.

Total Phenolics Measurement and Quantitative Analysis of Selected Components of LWDH Extracts
The total phenolics content of LWDH extracts was determined according to a modified Folin-Ciocalteu (FC) colorimetric method [41].Briefly, 40 mg of each LWDH extract was dissolved in 80% MeOH with 1% acetic acid, centrifuged to remove insoluble fraction and made into 1 mL sample solution.Twenty microlitres (mL) of this extract solution was dispensed in 96-well plate.To each well, 40 mL 10% FC reagent was added, mixed and incubated for 5 min in the dark at room temperature.Then 160 mL 700 mM sodium carbonate was added, the plate was covered with Parafilm and incubated in the dark at room temperature for 1.5 h.Finally, the absorbance at 750 nm was read on a SPECTRA max M2 plate reader (Molecular Devices Corporation, CA, USA).Gallic acid was used as a standard.A five-point standard curve was plotted in a linear range from 0.025 mg/mL to 0.200 mg/mL.Total phenolics content of samples is reported as the mean6SD of gallic acid equivalents (GAE) in mg per gram of dry material (mg GAE/g) from 3 replicated measurements.
To quantify the concentrations of major components in LWDH extracts, a HPLC-DAD method was used with separation being conducted on a TSK gel ODS-100 V column (4.66250 mm, 3 mm, Tosoh Bioscience) on Agilent HPLC 1100 system with DAD.LWDH extracts (10 mg) were accurately weighed and dissolved in 1 mL MeOH (for LWDH-EE) and water (for LWDH-WE).After 30 min of sonication the solution was filtered through 0.2 mm syringe filter and a 10 mL sample was injected into the system.For mobile phase, solvent A was water with 0.1% TFA and solvent B was acetonitrile with 0.1% TFA.Gradient was 3% B for 15 min, to 12% B at 17 min, and kept at 12% B for 16 min, then to 45% B at 39 min, with total run time of 87 min.Column temperature was set at 35uC, flow rate was 1 mL/min.The standards were accurately weighed and dissolved in methanol.Four different concentrations were used to make linear calibration curves.The linear ranges were at: 0.81 to 162.10 mg/mL for gallic acid; 0.83 to 333.30 mg/mL for 5-hydroxymethyl furfural.For sweroside, loganin, paeoniforin and paeonol, the linear range was at 0.83 to 166.67 mg/mL.The contents of these selected components in LWDH extracts with standards available from commercial sources were quantified and reported as mean6SD (mg/g) from 3 replicated analyses.

DPPH Radical Scavenging Assay
LWDH extract solutions prepared as described in section 2.3 were used for determination of DPPH radical scavenging activity using a 96-well plate method [42] with minor modification.Briefly, 10 mL of sample solution was added in each well followed by addition of 100 mL of 761.4 mM DPPH in 80% MeOH.The samples were incubated at room temperature in the dark for 2 h and the absorbance at 515 nm was read on a SPECTRA max M2 plate reader (Molecular Devices Corporation, CA, USA).Trolox was used as a standard.A seven-point standard curve was plotted from 0.25 mg/mL to 0.88 mg/mL.The results were expressed as mean6SD mmol of Trolox equivalents per gram of dry sample from 3 replicated measurements.

b-Amyloid (Ab) Aggregation Assay
The effect of LWDH extracts on Ab aggregation was determined using a thioflavin T (ThT) fluorescence assay as described previously [43].Briefly, 20 mL of b-amyloid 1-42 (25 mM in 10% DMSO/PBS ( 16)) was added to 2 mL sample (25 mg/mL in DMSO) in a 0.2 mL tube and mixed by gentle tapping.Tubes were covered to minimize sample evaporation and incubated in the dark at room temperature with no agitation.The extent of Ab aggregation was estimated by periodically aliquoting 10 mL of Ab1-42 in a 96-well plate.To each sample, 200 mL of 10 mM ThT in 0.1 M glycine buffer (pH 8.9) was added and the plate was read on a microplate reader (Varioskan, Thermo, USA) for fluorescence intensity at excitation of 450 nm and emission of 482 nm.All ThT fluorescence experiments were performed in triplicate and data expressed as mean 6 SD (N = 3).

LWDH Treatment to C. elegans
Stock solutions (100 mg/mL) of LWDH extracts were prepared in methanol (LWDH-EE) or water (LWDH-WE).The extracts were added to the nematode growth medium (NGM) to a final concentration of 0.1-2.0mg/mL of NGM using 0.05-0.1% methanol, just before plating.Gentamycin was added to the NGM at a concentration of 30 mg/mL to inhibit microbial contamination.The E. coli OP50 was spread on the NGM as food for C. elegans.Plain NGM or MeOH added to the NGM served as controls.The extract of Ginkgo biloba (EGb761) (1 mg/mL NGM) and in some experiments, quercetin (200 mg/mL NGM) was used as positive control.

Bioassays for b-amyloid-induced Paralysis
To determine if LWDH extracts suppress or delay the onset of b-amyloid induced progressive paralysis in CL4176 expressing muscle-specific Ab 1-42 [44], freshly laid eggs were transferred on to NGM containing LWDH extracts (water-LWDH-WE; ethanol-LWDH-EE) or controls and incubated for 36 h at 16uC.To initiate the amyloid-induced paralysis, the worms were up shifted from 16uC to 23uC.The scoring was performed at an hourly interval typically after 25 h at 23uC.The worms were scored as ''paralyzed'' based on either the failure of the worms to move their body with touch of a platinum loop, or the formation of a halo on bacterial lawn indicating a paralyzed condition.Each experiment was performed using at least 90 worms.The data represents mean of three different experiments (N = 270).
To determine the effect of LWDH-EE on deposition of amyloid plaques in C. elegans, we used transgenic CL2006 nematodes.Synchronous eggs (60-80) were transferred to NGM plates with or without LWDH-EE and incubated at 16uC for 3 days.The worms at late L4 stage (just before adulthood) were shifted into a 20uC environment for further development.Samples were harvested at different times (4 and 8 days after adulthood) and fixed overnight in 4% paraformaldehyde in PBS pH 7.4 at 4uC.The worms were then permeabilized by incubation for 24 h at 37uC in 5% bmercaptoethanol in 125 mM Tris, pH 7.4 containing 1% Triton X-100.Samples were washed 2-3 times with PBS-T, then mounted on glass slide and stained with 0.125% thioflavin-T in 50% ethanol for 2 min.The nematodes were destained with sequential washes with ethanol (50%, 75% and 90%) and observed under a fluorescent microscope (Olympus, Tokyo, Japan) for the presence of amyloid plaques in the head region.The experiment was repeated 2 times and the data expressed as mean6SE (N = 30) for each group.

Western Blotting of Ab Species
Worms were harvested in ddH 2 O containing protease inhibitor cocktail (16, Sigma), flash frozen in liquid nitrogen and stored at 280uC.Worms were boiled at 105uC for 10 min in a lysis buffer (62 mM Tris-HCl pH 6.8, 2% SDS (w/v), 10% glycerol (v/v), 4% b-mercaptoethanol (v/v) and 1X protease inhibitor cocktail), and then cooled on ice and centrifuged for 5 min at 14,000 g at 4uC.The protein in the supernatant was quantified using Bradford reagent (Biorad).Fifteen mg of protein was denatured prior to electrophoresis by boiling for 5 min in denaturation buffer (62 mM Tris-HCl pH 6.8, 2% SDS (w/v), 10% glycerol (v/v), 4% b-mercaptoethanol (v/v) and 0.0005% bromophenol blue (w/ v)).Samples were run at 140 V on 16% SDS BIS-Tris gel using Tris-glycine SDS running buffer (Biorad), for approximately 90 min.Ten to 175 kDa protein markers (Bioshop, Burlington Canada) were used as size references.The gel was transferred to 0.45 mM PVDF membrane (Immobilon P, Millipore) using 20% methanol TG buffer (Biorad), using 20 V for 120 min.
Blots were blocked in TBS-Tween +5% milk (100 mM Tris-7.5, 150 mM NaCl, 0.1% Tween-20 (v/v)).Amyloid protein species were detected with 6E10 (Covance) at 1:750 dilution; secondary anti-mouse IgG alkaline phosphatase conjugate (Sigma).Secondary alkaline phosphatase conjugate were developed using SigmaFast TM BCIPH/NBT tablets (Sigma) and the reaction stopped by addition of 20 mM EDTA (pH 8.5).Mean densities of b-amyloid bands were analyzed using Image-J software (National Institutes of Health, USA) on air dried blots.Equal loading was determined by replicate non-transferred Coomassie Blue stained, SDS-PAGE gel.The data is expressed as mean of 5 biological replicates using 4 and 20 kD bands.

In vivo Measurement of Reactive Oxygen Species (ROS) in C. elegans
Intracellular ROS was measured in treated and control C. elegans strain CL4176 using 2,7-dichlorofluorescein diacetate (H2DCF-DA) following a previously described method [26,30] with modifications.Briefly, freshly laid eggs (60-65 eggs per plate) were transferred to control or LWDH-EE (0.5 mg/ml) amended NGM plates and incubated for 36 h at 16uC.To initiate amyloid induced progressive paralysis, the worms were shifted to an incubator set at 23uC.The worms were harvested at 30 h after temperature shift using 500 mL of phosphate-buffered saline (PBS), washed twice with PBS to remove E. coli (OP-50) cells and transferred into 96-well plate (Costar) in a 200 mL volume of PBS containing Tween 20 (0.01%) and H2DCF-DA (final concentration 50 mM in PBS).The fluorescence was quantified in a Synergy HT microplate fluorescence reader (Bio-Tek Instruments, Winooski, VT) for 6 h at 37uC using the excitation at 485 nm and emission at 530 nm.Data represent mean6SE of three independent experiments expressed as percent fluorescence relative to MeOH control.

Fluorescence Microscopy of Reporter Gene Expression in LWDH-EE Treated C. elegans
To study the effect of LWDH extracts on HSP16.2 and SOD-3, C. elegans strains TJ375 (hsp-16.2::GFP) and CF1553 (sod3::GFP) were used.Synchronized TJ375 eggs were transferred on to NGM plates containing the extracts and incubated for 2 days at 20uC.For inducing heat shock response, L4 worms were shifted to a temperature controlled incubator set at 35uC for 2 h followed by recovery at 20uC before taking pictures at 24, 48 and 72 h.For SOD-3 response, pretreated L4 worms (CF1553) were transferred to new NGM plates containing 100 mM juglone and incubated overnight before analyzed with fluorescence microscopy.For quantification, the worms were anesthetized with sodium azide (10 mM) on agarose pad on a glass slide and the fluorescence was viewed under a microscope (Olympus, Japan) with excitations at 488 nm and emissions at 500-530 nm.The fluorescence intensity was analyzed using Image-J software.Each experiment was repeated twice and 15 worms per group were used in each experiment.Data represent mean6SE (N = 30) of two biological experiments.

Juglone Induced Stress in Wild Type N2
Wild type N2 worms were used to determine if LWDH-EE imparts tolerance to juglone induced oxidative stress.Adult N2 worms were allowed to lay eggs for 3 h on NGM plates.Freshly laid eggs were transferred to treatment plates containing LWDH-EE (0.5 mg/ml) or the MeOH control and allowed to develop up to late L4 stage (approximately 2.5 days) at 20uC.Gingko biloba extract (EGb761) served as the positive control.The pretreated L4 worms (30 worms/plate, 90 worms per group) were then transferred to NGM plates supplemented with juglone (300 mM).Survival of the worms was observed at hourly intervals until all the worms died in each of the treatment plates.The experiment was repeated two times and data represents mean6SE (N = 180) for each group.

Statistical Analysis
JMP IN software was used for statistical analyses.Comparison between treatments was done with unpaired Student's t test.For paralysis assays, a two-way ANOVA was used to determine significance.Differences at the p,0.05 level were considered significant and marked with an asterisk (*).

Identification and Quantification of Main Components in LWDH Extracts
As a classic TCM formula, LWDH has been well documented in TCM literature for improving cognition and the commercial products are regulated and standardized in China.A number of bioactive components are reported in the LWDH formula.However, the composition of products based on this complex formula may still vary to certain degree from one manufacturer to another.HPLC-based methods for quality control and component analysis have been applied to LWDH, and indeed, minor variations were shown to be evident [45].We first determined the main bioactive components in LWDH extracts used in our study with 1 H NMR and HPLC-MS analysis.In proton NMR, LWDH-WE and LWDH-EE are similar in terms of compound classes presented.However, higher sugar signals and lower levels of phenolics and other components were found in LWDH-WE, as compared to LWDH-EE (Figure S1).In HPLC analysis, C-18 column resolved six less polar components and their identities were revealed to be gallic acid, 5-hydroxymethyl furfural, sweroside, loganin, paeoniforin, and paeonol, based on available reference compounds and MS data (Figure S2 and Table S1).For high polarity components, a HILIC HPLC column was used generating the identity of loganin, gallic acid, glycosides of 5-hyrdoxymethyl furfural and gallic acid, monosaccharide monomers, dimers, and trimers as the main components based on ELSD and MS information (Figure S3 and Table S2).Additional monosaccharide dimers and trimers were found in LWDH-WE, as also indicated in the NMR spectra.The main components identified in this study are in general agreement with previously published reports [45][46][47].
Phenolic compounds such as gallic acid and paeonol in LWDH may contribute to its beneficial effects, so we quantified the total phenolics in LWDH extracts.By using Folin-Ciocalteu colorimetric method, the total phenolic contents of LWDH-WE and LWDH-EE extracts were determined to be 14.9760.36and 60.4363.56mg/g gallic acid equivalent, respectively.The results showed that ethanol extract contained higher concentration of phenolic components than the water extract, as revealed by NMR and HPLC analysis.
We further quantified the contents of 6 main components identified in LWDH extracts using HPLC-DAD method (Figure S4).5-Hydroxymethyl furfural content was determined to be 18.78860.071and 2.06560.004mg/g in LWDH-EE and LWDH-WE, respectively, the highest component measured for these extracts.Two phenolic compounds, gallic acid and paeonol, were 5.14460.168and 4.67560.004mg/g in LWDH-EE, while gallic acid was measured to be 0.77660.003mg/g in LWDH-WE, much lower than that of LWDH-EE.The other phenolic component, paeonol, however, was even not-detectable in LWDH-WE.Other components, including two iridoids loganin and sweroside, and paeoniforin were measured at 6.16760.025,0.78760.030,and 3.67660.210in LWDH-EE, and 0.85060.006,0.10460.004,and 0.48360.013mg/g in LWDH-WE, respectively.The ethanol extract not only showed to have higher content of phenolic compounds, but also contained specific compound such as paeonol that was not present in water extract.Significantly higher levels of other components in LWDH-EE may also be responsible for the observed differences in bioactivity between these two extracts.

DPPH Radical Scavenging Activity and Effect on Ab Aggregation of LWDH Extracts
We measured the antioxidant activity of LWDH extracts using DPPH radical scavenging assay.DPPH, 1,1-diphenyl-2-picrylhydrazyl, is a stable organic nitrogen radical with maximum UV-vis absorption at 515 nm.When it interacts with antioxidant, the solution color fades and this is monitored by a spectrometer.In our assay, antioxidant standard (+/2)-6-hydroxy-2,5,7,8-tetramethyl-chromane-2-carboxylic acid (Trolox) was used for the measurement.The results revealed that the ethanol extract LWDH-EE showed significantly higher antioxidant activity than the water extract LWDH-WE.The radical scavenging activities of LWDH-WE and LWDH-EE were 102.2065.52 and 402.76618.52mmol Trolox equivalent (TE)/g, respectively.The high DPPH activity of ethanol extract correlated with higher phenolic content of LWDH-EE.
Ab aggregation, fibril formation and deposition are believed to cause neurotoxicity and eventually the onset of neurodegenerative diseases, such as AD [48,49].Polyphenols and other types of small molecules are reported to be potential inhibitors of Ab aggregation [43,[50][51][52], suggesting this to be, at least in part, mechanisms of action for prevention and treatment of AD.By using the ThT fluorescence method and curcumin as a positive control [53,54], we determined the effect of LWDH extracts on Ab aggregation.However, LWDH extracts did not affect Ab aggregation, suggesting that LWDH extracts appears not to modify or intervene in the Ab aggregation process, especially in vitro.

Ab-induced Paralysis in the Transgenic C. elegans is Alleviated by LWDH Extracts
We used C. elegans strain CL4176 expressing Ab1-42 [55] to determine the effects of LWDH on Ab-induced toxicity in the whole animal.The results showed that LWDH-EE was highly effective as compared to LWDH-WE in protecting C. elegans against b-amyloid induced toxicity (Figure 1).C. elegans treated with ethanol extract of LWDH .0.1 mg/mL delayed the onset of paralysis compared to control (MeOH).This effect was not significantly different between 0.5-2 mg/mL concentrations of LWDW-EE tested (p.0.05) (Figure 1A).LWDH-EE treated worms showed paralysis after 30 h while the onset of paralysis started at 25 h in the MeOH control and more than 50% of worms were paralyzed by this time (Figure 1A).In contrast, LWDH-WE effect was not as strong as LWDH-EE where the paralysis phenotype appeared by 27 h after temperature induction.The higher dosage (1-2 mg/mL) of LWDH-WE showed an increasing effect in delaying the paralysis phenotype than NGM control but this effect was not as strong as LWDH-EE (0.5-2 mg/ mL) that showed higher efficiency (p,0.05)versus the control in delaying paralysis.When compared with positive control, EGb761 (1 mg/mL), the effect of LWDH-WE (1-2 mg/mL) was similar to the Ginkgo extract (p.0.05) (Figures 1A & 1B).Quercetin (200 mg/mL) a potent antioxidant, did not negate paralysis.These observations suggest that LWDH and more specifically LWDH-EE may have the potential to protect against b-amyloid induced toxicity.
To determine if the effect of LWDH on delayed onset of paralysis was associated with a decrease in Ab accumulation in transgenic CL4176, we performed Western blot analysis of LWDH-EE treated worms sampled at 25 h after temperature upshift (Figure 2A).The visual observation of immunoblot developed with anti-Ab monoclonal antibody 6E10 did not show significant difference in monomeric or oligomeric Ab species.The quantitative analysis of Ab bands with ImageJ showed a small reduction in Ab proteins (4 and 20 kD) but this difference was not significant (Figure 2B).
When we tested the effectiveness of LWDH-EE on C. elegans strain CL2006, which constitutively express Ab, a marginal delay in paralysis was observed (data not shown).Further, b-amyloid deposits were detected in the head region of control and treated worms (Figure 2C).ImageJ analysis showed that LWDH-EE reduced b-amyloid deposits in the worms by day 4 after adulthood, the effect of LWDH-EE however diminished with time as the difference was no longer significant by the 8 th day.Quercetin used in this experiment as positive control also failed to suppress b-amyloid deposition.These results seem to indicate that the protective effect of LWDH against b-amyloid induced toxicity may be, in part, due to the delayed Ab accumulation and deposition in the worms that contributes to delayed onset of paralysis phenotype.

Ethanol Extract of LWDH Reduces Oxidative Stress in C. elegans
LWDH extracts were shown to be potent in scavenging DPPH radical in vitro.To determine whether LWDH extract has effect in reducing reactive oxygen species (ROS) in C. elegans, we investigated the in vivo ROS activity in LWDH-EE treated and control worms using transgenic strain, CL4176.LWDH-EE reduced ROS in the worms as compared to control.This was evident by a highly significant reduction (p,0.001) in mean fluorescence detected during 6 h period (Figure 3A).The results showed that LWDH-EE suppressed ROS by more than 50%.This response was similar to the effect of a well known antioxidant EGb 761.These results suggest that the delayed onset of Ab related paralysis phenotype in CL4176 worms with LWDH treatment might be, in part, due to the ROS suppressing properties of the LWDH extract.
We further observed that the LWDH-EE also protected wild type N2 against oxidative stress generated by juglone.The worms pre-incubated in LWDH-EE (0.5 mg/mL) were significantly protected against juglone (300 mM) induced oxidative stress (Figure 3B).The mean survival of the worms was significantly higher (40%) (N = 180, p,0.05) with LWDH-EE at 6 h after the stress compared to the control (Figure 3B).The T50 (time to 50% mortality) was observed at 3 h for control and 5.8 h for the LWDH-EE treated worms (p,0.05).The survival of the worms with quercetin treatment was significantly higher than LWDH-EE and EGb761 at 7 h.Although the survival of the worms with LWDH-EE treatment during juglone induced oxidative stress was higher than that of EGb761, this difference was, however, not significant (p.0.05).

Gene Expression Analysis of LWDH-EE Treated C. elegans
To relate the effect of LWDH-EE on the molecular response in C. elegans, the transcript levels of three genes, amy-1, sod3 and hsp16.2 were studied in transgenic C. elegans strain CL4176 after temperature upshift.Quantitative Real-Time PCR revealed differential expression of these genes after temperature upshift (Figure 4).The results showed that the transcripts of all three genes (amy-1, sod3 and hsp16.2) were initially higher in LWDH-EE treatment than control at 10 h after temperature upshift.However, amy-1 and sod-3 transcript abundance was less in LWDH treated worms as compared to the control at 15 h after the temperature upshift.Interestingly, the hsp16.2transcript, which was higher at 10 h, further increased to more than six fold (p,0.05) with LWDH-EE treatment as compared to the control.

LWDH-EE Increases hsp-16.2::GFP Expression in C. elegans
LWDH induced stress response in transgenic C. elegans strain TJ375 carrying hsp16.2::GFPreporter was determined after 2 h thermal stress (35uC) and overnight recovery at 20uC.The GFP fluorescence quantified in the head region of the worms showed a remarkably higher (p,0.05)hsp-16.2::GFPexpression after heat shock with LWDH-EE compared to the LWDH-WE or untreated control worms (Figure 5).The LWDH-EE-specific effect remained evident even at day 3 post thermal stress with almost twice the expression (p,0.05)than other treatments.These results are in agreement with the gene expression results from C. elegans strain CL4176 described above, and suggests that LWDH-EE treated worms might be protected from Ab toxicity due to an up regulation of HSP16.2.

LWDH-EE Affects the Expression of sod3::GFP in C. elegans
The molecular response in C. elegans treated with LWDH-EE under oxidative stress was further studied using transgenic worms (CF1553) expressing sod3::GFP.The control or LWDH-EE treated L4 worms were subjected to overnight exposure to 100 mM juglone.Interestingly, relative fluorescence of sod3::GFP in different biological replicates were only 5-10% higher in LWDH-EE treated worms at normal conditions (no juglone stress) compared to control worms the difference was however not significant (Figure 6).Similar pattern was observed with juglone treatment as the increase in fluorescence intensity was not significant in LWDH-EE treated worms compared to control worms (Figure 6).It appears that the tolerance to juglone induced stress in the LWDH-EE treated worms might involve additional molecular responses along with SOD-3.

Discussion
Alzheimer's disease (AD) is a neurodegenerative disease associated with ageing [56].Besides extensive pre-clinical and clinical research on pathology and etiology of this disorder, other than symptomatic treatment strategies for treating this debilitating disease are used, the cause and a cure for AD have yet to be fully elucidated and achieved.The future success of the treatment and management of AD is therefore highly dependent on early detection and treatment with drugs that block, mitigate, slow, or suppress any of the causative and/or putative disease-associated co-factors and delay the onset of AD.Traditional Chinese medicine, along with other natural product-based therapies have been used in preventing or treating age-related deterioration in cognitive or memory performance [57][58][59], and a number of studies have been conducted to examine their potential benefits in AD.
Liuwei Dihuang (LWDH), a classic six-herb TCM formula, has been used for over a thousand years to improve or restore declined functions related to aging process and geriatric diseases, such as impaired mobility, vision, hearing, cognition and memory [1].Several studies showed that LWDH can modulate neuronal and synaptic function that contributes to the cognitive enhancement  We examined if LWDH has in vitro and/or in vivo effect on bamyloid based on experimental evidences from Ab aggregation, expression and/or deposition.Although, LWDH extracts did not reduce aggregation of amyloid protein in vitro, LWDH extracts were found to be effective in vivo and we observed a delayed onset of b-amyloid induced paralysis in C. elegans strain CL4176 expressing Ab peptide [21] (Figure 1).When examined for toxic amyloid plaques in C. elegans strain CL2006, b-amyloid deposits were initially low with quercetin and LWDH-EE treatments (Figure 2), the difference however diminished with age.Gene expression study demonstrated that LWDH suppressed amy1 in C. elegans strain CL4176 suggesting that the treatment did not have a significant effect on the transcription of amy1 gene.Ab transgene expression has not always correlated with b-amyloid deposition in the worms expressing Ab1-42 [30,35,37].One possible explanation is the differences in transgenic strains used in the experiments and the time of sampling the worms for analysis.During initial (at 10 h) observations, we also noted upregulation of amy1 gene in CL4176 with LWDH-EE treatment, however there was repression of amy1 gene at 15 h after temperature upshift (Figure 4).Results suggest that delayed onset of the paralysis was at least in part due to reduced amy1 transcription leading to delay in b-amyloid deposition during the temperature upshift.It remains to be determined if LWDH-EE treatment delayed paralysis correlate with differential oligomerization of Ab species.
To determine potential mechanisms of LWDH activity in the worm, we examined whether it affected the oxidative stress response in C. elegans.LWDH possesses strong antioxidant activity [60,61] that might be one of the contributing factors for the delayed onset of paralysis in the worms.LWDH-EE offered significant protection against oxidative stress in C. elegans as evidenced by results of our in vivo experiments.Oxidative stress is involved in AD pathogenesis and accumulation of ROS along with the deposition of toxic amyloid species is proposed to exacerbate the condition in AD patients [62,63].The antioxidant-based therapies have been suggested as a potential avenue to mitigate AD as has been demonstrated using an AD mouse model [64,65].In C. elegans, oxidative stress strongly correlates with Ab toxicity [66] and it is well known that a number of natural products reduce reactive oxygen species which are shown to be neuroprotective [31].Our results support the hypothesis that ROS reducing activity of LWDH might be one of the mechanisms by which it protected C. elegans against Ab toxicity.Strong ROS scavenging activity did not, however, reduce Ab accumulation in LWDH treated worms suggesting that formation of toxic pre-fibrillar Ab species [66] could have been reduced in LWDH treated CL4176 worms.
Small heat shock proteins (HSPs) are collection of low molecular weight polypeptides with chaperone-like activity that increase survival of C. elegans under stress and are induced with Ab expression [44].Since heat shock response in C. elegans is a neuronally-controlled behavior [67], induction of HSP-16 by Ab is suggested to be a protective response to the abnormal accumulation of toxic proteins [68].Previous reports showed that in vivo bamyloid peptide toxicity was suppressed by over expression of HSP-16.2 in C. elegans [68].The LWDH treated worms showed significantly higher expression of heat shock proteins as determined through analysis of hsp16.2 transcript abundance and GFP reporter expression.The GFP expression was elevated even up to 3 days after heat shock suggesting that HSP-16.2 was involved in the protection against Ab induced toxicity by LWDH.Our results are in close agreement to previous reports and suggest that LWDH might play role in increasing the expression of this small heat shock  protein in C. elegans which might contribute to suppression of or delayed the onset of paralysis in the treated animals.Mechanistically, this could be possible through enhanced plaque clearance but cellular distribution of Ab or modulations of multimerization pathways of Ab that reduce the formation of toxic oligomer species, could also be speculated and worthy of further examination [68].
In summary, LWDH extracts, particularly the ethanol extract (LWDH-EE) alleviates b-amyloid induced paralysis in C. elegans through increasing heat shock proteins (HSP16.2),lowering amy1 and reducing ROS, all of which could have mutually exert the protective mechanisms against b-amyloid toxicity.The finding that b-amyloid induced paralysis was alleviated in C. elegans AD model potentially provide more insights into the action of LWDH as a potential neuroprotective agent and warrants further testing to be conducted using higher order experimental models.

Figure 1 .
Figure 1.LWDH delayed b-amyloid induced paralysis in transgenic C. elegans, strain CL4176.Time course of Ab-induced paralysis in the transgenic CL4176 strain treated with LWDH ethanol (A), and water (B) extract.Egb761 and quercetin served as positive controls whereas untreated NGM or MeOH +NGM were used as controls.C. elegans paralysis phenotype was monitored 20 h following temperature upshift to 23uC and expressed as percent survival of the worms drawn based on three individual experiments with 90 worms in each group (N = 270).The significances of differences in paralysis curves were p,0.05 versus the control.doi:10.1371/journal.pone.0043990.g001

Figure 2 .Figure 3 .
Figure 2. LDWH reduced b-amyloid species in C. elegans. A. Western blot of Ab species in the transgenic C. elegans CL4176 with and without LWDH-EE treatment.The CL4176 proteins were loaded on 16% acrylamide gel and immunoblotted with an anti-Ab antibody (6E10).Lanes 1 and 2 were loaded with CL4176 proteins from 16uC whereas lane 3 (MeOH control) and lane 4 (LWDH-EE) were loaded with protein extracted from worms incubated in permissive temperature.The blot represents five independent experiments.B. Quantification of Ab species (4 and 20 kD) using ImageJ software.Data are expressed as mean density of two indicated bands6SE based from five independent experiments.C. Ab species in CL2006 determined by Thioflavin-T stain.CL2006 worms were treated with LWDH-EE and stained with ThT and viewed for amyloid aggregation in the head region anterior to the pharyngeal bulb.All ThT fluorescence experiments were performed two times.Data were obtained from two biological experiments with 15 worms in each group (N = 30) and expressed as b-amyloid deposits relative to control worms.Error bars represent SEM.*p,0.05.doi:10.1371/journal.pone.0043990.g002

Figure 4 .
Figure 4. Gene expression in transgenic C. elegans, strain CL4176.Quantitative real-time PCR results of LWDH-EE treatment on the expression of the amy1, sod3 and hsp16.2 in C. elegans strain CL4176.Error bars indicate 6SE.C. elegans were raised on NGM or NGM containing LWDH-EE at 16uC and then shifted after 36 h to 23uC.The worms (,80-100) were harvested at 20 h after temperature upshift for RNA extraction.The relative gene expression was determined with 2 2DDCt method.The gene ama-1 was used as the internal control.Data were average from two independent runs (*p,0.05).doi:10.1371/journal.pone.0043990.g004

Figure 6 .
Figure 6.Effect of LWDH on the expression of sod3::GFP in C. elegans.To determine role of SOD-3 in LWDH induced response of C. elegans, L4 worms of CF1553 previously treated from egg stage with LWDH-EE were exposed to 100 mM juglone overnight and then observed for the fluorescence.The fluorescence intensity was analyzed using Image-J software (National Institutes of Health, USA).The images were quantified based on GFP intensity (mean6SE) in CF1553 from two independent experiments with 15 worms in each experiment (N = 30, *p,0.05).doi:10.1371/journal.pone.0043990.g006