Lactucopicrin ameliorates oxidative stress mediated by scopolamine-induced neurotoxicity through activation of the NRF2 pathway

Accepted Manuscript Lactucopicrin ameliorates oxidative stress mediated by scopolamine-induced neurotoxicity through activation of the NRF2 pathway Ramu Venkatesan, Lalita Subedi, Eui-Ju Yeo, Sun Yeou Kim PII:

S0197-0186(16)30172-3

DOI:

10.1016/j.neuint.2016.06.010

Reference:

NCI 3888

To appear in:

Neurochemistry International

Received Date: 29 December 2015 Revised Date:

13 June 2016

Accepted Date: 20 June 2016

Please cite this article as: Venkatesan, R., Subedi, L., Yeo, E.-J., Kim, S.Y., Lactucopicrin ameliorates oxidative stress mediated by scopolamine-induced neurotoxicity through activation of the NRF2 pathway, Neurochemistry International (2016), doi: 10.1016/j.neuint.2016.06.010. 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 proof before it is published in its final 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.

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT Lactucopicrin

ameliorates

oxidative

stress

mediated

by

scopolamine-induced

neurotoxicity through activation of the NRF2 pathway

1

Ramu Venkatesan1, Lalita Subedi1, Eui-Ju Yeo4, and Sun Yeou Kim1,2,3*

2 3

1

4

2

Gachon Medical Research Institute, Gil Medical Center, Inchon 21565, Republic of Korea

5 6

3

Gachon Institute of Pharmaceutical Science, Gachon University; #191 Hambakmoe-ro, Yeonsu-gu, Incheon 21936, Republic of Korea

7 8

4

Department of Biochemistry, College of Medicine, Gachon University, #191 Hambakmoero, Yeonsu-gu, Incheon 21936, Republic of Korea

SC

RI PT

Lab of Pharmacognosy, College of Pharmacy, Gachon University, #191, Hambakmoero, Yeonsu-gu, Incheon 21936, Republic of Korea

M AN U

9 10

*Corresponding author. Sun Yeou Kim; Lab of Pharmacognosy, College of Pharmacy, Gachon University, #191, Hambakmoero, Yeonsu-gu, Incheon 21936, Republic of Korea.

AC C

EP

TE D

Tel.: +81-32-899-6411; Fax.: +82-32-899-8962; [email protected]

ACCEPTED MANUSCRIPT Abstract

12

Cholinergic activity plays a vital role in cognitive function, and is reduced in individuals with

13

neurodegenerative diseases. Scopolamine, a muscarinic cholinergic antagonist, has been

14

employed in many studies to understand, identify, and characterize therapeutic targets for

15

Alzheimer's disease (AD). Scopolamine-induced dementia is associated with impairments in

16

memory and cognitive function, as seen in patients with AD. The current study aimed to

17

investigate the molecular mechanisms underlying scopolamine-induced cholinergic neuronal

18

dysfunction and the neuroprotective effect of lactucopicrin, an inhibitor of acetylcholine

19

esterase (AChE). We investigated apoptotic cell death, caspase activation, generation of

20

reactive oxygen species (ROS), mitochondrial dysfunction, and the expression levels of anti-

21

and pro-apoptotic proteins in scopolamine-treated C6 cells. We also analyzed the expression

22

levels of antioxidant enzymes and nuclear factor (erythroid-derived 2)-like 2 (NRF2) in C6

23

cells and neurite outgrowth in N2a neuroblastoma cells. Our results revealed that 1 h

24

scopolamine pre-treatment induced cytotoxicity by increasing apoptotic cell death via

25

oxidative stress-mediated caspase 3 activation and mitochondrial dysfunction. Scopolamine

26

also downregulated the expression the antioxidant enzymes superoxide dismutase,

27

glutathione peroxidase, and catalase, and the transcription factor NRF2. Lactucopicrin

28

treatment protected C6 cells from scopolamine-induced toxicity by reversing the effects of

29

scopolamine on those markers of toxicity. In addition, scopolamine attenuated the secretion

30

of neurotrophic nerve growth factor (NGF) in C6 cells and neurite outgrowth in N2a cells. As

31

expected, lactucopicrin treatment enhanced NGF secretion and neurite outgrowth. Our study

32

is the first to show that lactucopicrin, a potential neuroprotective agent, ameliorates

33

scopolamine-induced cholinergic dysfunction via NRF2 activation and subsequent expression

34

of antioxidant enzymes.

35

Key words: Lactucopicrin, Scopolamine, ROS, NRF2, Antioxidant, AChE inhibitors

AC C

EP

TE D

M AN U

SC

RI PT

11

ACCEPTED MANUSCRIPT Chemical Compounds:

37

Lactucopicrin (PubChem CID: 174880); Galantamine hydrobromide (PubChem CID:

38

121587); (-)-Scopolamine hydrobromide trihydrate (PubChem CID: 20055509); Retinoic

39

acid (PubChem CID: 444795); 2′,7′-Dichlorofluorescein diacetate (PubChem CID: 104913);

40

Rhodamine 123 (PubChem CID: 65217); Thiazolyl Blue Tetrazolium Bromide (PubChem

41

CID: 64965)

RI PT

36

42

Abbreviations:

44

ROS, reactive oxygen species; AD, Alzheimer's disease; AChE, acetylcholine esterase; CNS,

45

central nervous system; NGF, neurotrophic nerve growth factor; ∆ψm, mitochondrial

46

membrane potential; SOD, superoxide dismutase; GPx, glutathione peroxidase; NRF2,

47

nuclear factor (erythroid-derived 2)-like 2; DCFH-DA, 2',7'-dichlorofluorescin diacetate;

48

FBS, fetal bovine serum; mAChR, muscarinic acetylcholine receptor; FITC, fluorescein

49

isothiocyanate; PI, propidium iodide; ANOVA, analysis of variance; P, probability; F, degree

50

of freedom; BcL-2, B-cell CLL/lymphoma 2; Bax, BcL-2-associated X Protein; N2a cell,

51

mouse neuroblastoma cell; C6 cell, rat glioma cell; ERK, extracellular signal-regulated

52

kinase; GSK-3β, glycogen synthase kinase-3β; BcL-xl, B-cell lymphoma extra-large; Bad,

53

Bcl-2-associated death promoter; Cyto C, cytochrome C

AC C

EP

TE D

M AN U

SC

43

ACCEPTED MANUSCRIPT 54

Introduction Cholinergic neuronal activity plays a prominent role in the basal forebrain and is

56

essential for cognitive functions, such as learning and memory. Cognitive impairment is

57

negatively correlated with levels of acetylcholine in the cerebrospinal fluid of patients with

58

dementia (Tohgi et al., 1996). Acetylcholine receptor-deregulating compounds such as

59

scopolamine have been used to create AD models for studies into the pathophysiological

60

mechanisms of AD and drug development (Hasselmann, 2014). Scopolamine, from Giovanni

61

Scopoli, is a broad-spectrum, non-specific, and well-known cholinergic antagonist with high

62

binding affinity for muscarinic receptors (Konar et al., 2011). Bajo et al. and others reported

63

that treatment of scopolamine might induce AD. Therefore it has been used to develop in

64

vitro and in vivo AD models (Bajo et al., 2015; Lee et al., 2014; Moosavi et al., 2012;

65

Pandareesh and Anand, 2013; Weinreb et al., 2012).

M AN U

SC

RI PT

55

AD is the most common neurodegenerative disease. It is characterized by reduced

67

neuronal activity and loss of neuronal cells, which results in memory impairment (Ghezzi et

68

al., 2013). Memory impairment becomes more severe with age and has a major impact on

69

daily activities, which negatively affects the quality of life of afflicted individuals and their

70

family members (Brookmeyer et al., 2007). Neuronal activity can be controlled by metabolic

71

support, extracellular ionic balance, synaptic transmission, and neurotrophic factors, all of

72

which have been implicated in memory function (León et al., 2013; Pandareesh and Anand,

73

2013). AD is one of complex neurodegenerative disorders. Only neuron-oriented research

74

can't overcome the Alzheimer's disease. Besides the degeneration of cholinergic neurons, it is

75

essential to understand the role glial and microglia cells in the AD pathogenesis. Recently,

76

Dzamba. D et al. reported that glial cells must be the key elements of Alzheimer´s disease

77

(Dzamba et al., 2016). Therefore, our attention towards glia would serve as important

78

momentum to turn the significance of glia in the pathogenesis and progression of AD.

AC C

EP

TE D

66

ACCEPTED MANUSCRIPT Memory impairment caused by neuronal cell loss can result from a variety of factors,

80

such as beta amyloid (Aβ) (Blennow et al., 2015), free radicals, pesticides (Li et al., 2015;

81

Yegambaram et al., 2015), and malnutrition (Ogawa, 2014). These factors are generally

82

accepted as part of the etiology of AD, particularly when they affect the cerebral cortex and

83

other areas of the brain (Kása et al., 1997). The extent of cholinergic neuron loss in the

84

central nervous system (CNS) correlates with the severity of cognitive impairment.

85

Scopolamine treatment generates reactive oxygen species (ROS) (Tao et al., 2014). Oxidative

86

stress-induced cell death is an important cause of neurodegeneration in this AD model.

87

Changes in the expression levels of synaptic proteins, neurotrophic factors, and antioxidant

88

enzymes may be responsible for scopolamine-induced neurotoxicity, and modulation of such

89

factors may be a way to protect cells or restore them to their original state (Xiong et al., 2015).

90

Recent studies showed that acetylcholine esterase (AChE) inhibitors suppress

91

synaptic dysfunction, Aβ plaque formation, and other causes of neuronal damage, such as

92

inflammatory reactions caused by T-cell activation, cytokines, and CNS inflammation (Kim

93

et al., 2014b; León et al., 2013). AChE inhibitors, such as donepezil, rivastigmine, and

94

galantamine, are approved for the treatment of AD patients (Murray et al., 2013). These

95

inhibitors bind to and reversibly inactivate cholinesterase and inhibit hydrolysis of

96

acetylcholine, resulting in increased acetylcholine concentration in cholinergic synapses,

97

which improves synaptic function. However, synthetic AChE inhibitors are associated with a

98

number of adverse events, such as nausea, diarrhea, and vomiting, which has contributed to

99

discontinuation in Phase II/ III clinical trials and extremely cautious use in patients with

100

underlying conditions. The side effects of synthetic AChE inhibitors manifest as salivary

101

gland dysfunction, arrhythmias, gastrointestinal disorders, hepatotoxicity, miosis, respiratory

102

problems, bradycardia, bronchospasm, and cardiac diseases (Godyń et al., 2016; Haerter and

103

Eikermann, 2016; Kim et al., 2014b). Many natural plant sources have been shown to have

AC C

EP

TE D

M AN U

SC

RI PT

79

ACCEPTED MANUSCRIPT neuroprotective effects. Moreover, the side effects of natural compounds are often not as

105

serious as those of synthetic drugs. Natural compounds also have strong antioxidant and

106

neurotrophic-mimetic activities and many other properties that make them preferable to

107

synthetic compounds (Kim et al., 2014b). In an effort to understand the mechanism of

108

scopolamine-induced toxicity in astrocytes, we used a reversible AChE inhibitor,

109

lactucopicrin (also known as intybin), a natural sesquiterpene lactone derived from Lactuca

110

virosa (wild lettuce), Cichorium intybus, and dandelion coffee. Lactucopicrin-containing

111

plants have been used as antimalarial agents, sedatives, and analgesics in humans (Rollinger

112

et al., 2005; Wesołowska et al., 2006).

SC

RI PT

104

In the current study, we examined the neuroprotective effects of lactucopicrin against

114

scopolamine-induced neurotoxicity in C6 glioma cells, with a focus on ROS production and

115

mitochondrial dysfunction as assessed via cytochrome C release and mitochondrial

116

membrane potential (∆ψm), free radical scavenging antioxidant enzymes such as superoxide

117

dismutase (SOD), glutathione peroxidase (GPx), and catalase, and NGF secretion. We also

118

investigated the neurodifferentiation effect of scopolamine on N2a neuroblastoma cells. In

119

addition, the molecular mechanisms underlying the effects of lactucopicrin were addressed. A

120

recent study showed that nuclear factor (erythroid-derived 2)-like 2 (NRF2), a transcription

121

factor that induces endogenous defense pathways and the generation of antioxidant enzymes

122

(Ni et al., 2014), is involved in protection against oxidative stress in a model of AD. In the

123

present study, we examined the ability of lactucopicrin to attenuate oxidative stress in a

124

scopolamine-induced AD model via regulation of NRF2.

AC C

EP

TE D

M AN U

113

125 126

2. Materials and Methods

127 128

2.1. Cell lines and reagents

ACCEPTED MANUSCRIPT C6 glioma and N2a neuroblastoma cells were obtained from the Korean Cell Line

130

Bank (Seoul, South Korea). Hyclone, 2',7'-dichlorofluorescin diacetate (DCFH-DA), MTT

131

[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide], retinoic acid, scopolamine,

132

and rhodamine 123 were purchased from Sigma-Aldrich (St. Louis, MO, USA).

133

Lactucopicrin was purchased from Extrasynthese (Lyon Nord-69730, Genay, France) and

134

galantamine hydrobromide was purchased from Tocris (Bristol, BS11 0QL, UK). All other

135

chemicals were taken from our laboratory stocks.

137

SC

136

RI PT

129

2.2. Cell culture and treatments

C6 rat glioma cells were grown in high-glucose DMEM medium supplemented with 1%

139

penicillin/streptomycin and 10% heat-inactivated fetal bovine serum (FBS) in a humidified

140

incubator under 5% CO2 and 95% air at 37°C and maintained by changing media every other

141

day. Cells at 60% confluence were pre-treated with scopolamine (3 mM) in serum-free

142

DMEM medium for 1 h. The medium was then replaced by DMEM medium containing 2%

143

FBS and vehicle, lactucopicrin (0.5, 1, or 2 µM), or galantamine (2 µM) for 24 h, at which

144

point cells were subjected to experimental assays.

147

TE D

EP

146

2.3. Cell viability assay

AC C

145

M AN U

138

An MTT assay was used to assess the viability of C6 cells. C6 cells grown to a

148

density of 10×104 in 24-well plates were exposed to various concentrations (1, 3, 5, or 10

149

mM) of scopolamine and assessed for viability after 24 h. Because scopolamine induced

150

cytotoxicity in a dose-dependent manner, we selected 3 mM as the dose of scopolamine for

151

the cytoprotection assay with lactucopicrin and galantamine. Cells were pre-treated with 3

152

mM or 5 mM scopolamine for 1 h and then treated with various concentrations (0.5, 1, or 2

153

µM) of lactucopicrin or 2 µM galantamine for 24 h. The culture media was collected and

ACCEPTED MANUSCRIPT stored at -20°C. The cells were incubated with MTT reagent (0.5 mg/ml final concentration)

155

for 1 h at 37°C. The MTT solution was then removed and formazan was dissolved by

156

addition of DMSO. The concentration of solubilized blue formazan was measured at 570 nm

157

using an ELISA reader.

158 159

2.4. Measurement of intracellular ROS

RI PT

154

To quantify the levels of intracellular ROS, C6 cells were seeded in 24-well plates

161

with sterilized coverslips and treated with lactucopicrin or galantamine as described above.

162

After 24 h treatment, 30 µM DCFH-DA was added to the culture medium for 30 min in a

163

CO2 incubator as described previously (Lee et al., 2007). Cells were then washed three times

164

with PBS and suspended in 200 µl of PBS. DCF fluorescence was observed using a

165

fluorescent microscope, and DCF fluorescence density was estimated from the images that

166

were acquired using a Juli smart fluorescence cell analyzer microscope. Percent ROS

167

generation was calculated from DCF fluorescence intensity and is shown as the mean ±

168

standard deviation of at least three experiments.

M AN U

TE D

170

2.5. Determination of ∆ψm

EP

169

SC

160

To determine the mitochondrial membrane potential (∆ψm), C6 cells were seeded in

172

6-well plates with sterilized coverslips and treated with lactucopicrin or galantamine as

173

described above. After 24 h treatment, cells were fixed with 4% paraformaldehyde for 20 min

174

at room temperature, and then washed with PBS three times at room temperature. Cells were

175

then treated with rhodamine 123 (50 nM) and kept in the dark for 30 min as per (Wang et al.,

176

2011). Coverslips were mounted on slides using mounting solution and cells were examined

177

using a confocal laser scanning microscope (Nikon A1+, Japan) with an excitation

AC C

171

ACCEPTED MANUSCRIPT 178

wavelength of 511 nm and an emission wavelength of 534 nm. Change in fluorescence

179

intensity reflects ∆ψm and was assessed by comparing experimental with control cells.

180 181

2.6. Evaluation of neurite outgrowth Lactucopicrin was evaluated for its effect on neurite outgrowth in N2a cells. N2a cells

183

were seeded at a density of 5×104 per well in 24-well plates and treated with lactucopicrin

184

and galantamine for 24 h, as detailed above. Images were captured using an Essen IncuCyte

185

ZOOM v2013B Rev1 (Essence Bioscience Inc., Ann Arbor, MI, USA). Retinoic acid and

186

galantamine were used as positive controls for neurite outgrowth. The collected images were

187

used to measure neurite length, and percent neurite outgrowth was calculated and plotted

188

(Moon et al., 2014).

189

2.7. Quantification of soluble NGF

TE D

190

M AN U

SC

RI PT

182

To estimate the level of soluble NGF, C6 cells were seeded in 24-well plates and

192

treated with lactucopicrin and galantamine as described above. After 24 h treatment, the

193

supernatant was stored at -20 °C until analysis of the NGF level (Kang et al., 2011). NGF

194

quantification was performed as described in the protocol supplied by the manufacturer of the

195

β-NGF ELISA kit (R&D system, Minneapolis, MN). NGF levels are expressed in pg/ml and

196

galantamine was used as a positive control.

198

AC C

197

EP

191

2.8. Western blotting

199

C6 cells (10×104) were seeded in a 100-mm dish and drug treatment was performed

200

as described above. After 24 h treatment, cells were harvested and homogenized in RIPA

201

lysis buffer containing PMSF, aprotinin, and protease inhibitor cocktail for 30 min at 4°C.

ACCEPTED MANUSCRIPT Protein concentration was assessed using Bradford's method (BioRad DC). 25-35 µg of

203

protein was separated using 12% SDS-PAGE and transferred onto a nitrocellulose membrane.

204

The membranes were blocked with 5% skim milk for 1 h, washed with TBST 3 times, and

205

incubated overnight with primary antibodies in 5% skim milk at 4°C. The membranes were

206

then washed with TBST buffer 3 times and then incubated with secondary antibody (1: 5,000)

207

in 5% skim milk for 1 h at room temperature. Protein bands were visualized using an ECL

208

detection system and band intensity was quantified by ImageQuant TL software v1.8.0.0 (GE

209

Healthcare Life Sciences). Protein levels were normalized to α-tubulin.

SC

RI PT

202

211

2.9. Determination of SOD activity

M AN U

210

SOD activity was studied using water-soluble tetrazolium salt (WST-1) (Yuhai and

213

Zhen, 2015). C6 cells (10×104) were seeded in 60 mm dishes and incubated overnight. Cells

214

were treated with scopolamine and lactucopicrin as detailed above. After 24 h treatment, the

215

cells were harvested in PBS using a cell scraper and sonicated. The cell lysate was

216

centrifuged at 3,000 rpm for 5 min, and then protein levels were quantified using Bradford's

217

method (BioRad DC). Cell lysates (50 µg protein/ml) were subjected to a SOD activity assay

218

according to the manufacturer’s protocol (Dojindo Laboratories, Kumamoto, Japan).

220 221

EP

AC C

219

TE D

212

2.10. Determination of catalase activity A catalase assay was carried out as previously described (Aebi, 1984). C6 cells

222

(10×105) were seeded in 60 mm dishes and treated with drugs as detailed above. Cell

223

homogenates were prepared as described in the user manual of the assay kit (BioVision, CA

224

95035, USA). Cell lysates were stored at -80°C until quantification. The experiment was

225

performed in 96-well plates using the OxiRed probe from the catalase assay kit (Chen et al.,

ACCEPTED MANUSCRIPT 226

2014a), and absorbance was measured at 570 nm. The results are expressed as micromoles of

227

hydrogen peroxide (H2O2) decomposed per minute per milligram of protein.

228 229

2.11. Determination of GPx activity GPx activity was measured using a previously described method (Kang et al., 2014).

231

C6 cells were seeded in 60 mm dishes and drug treatment was performed as detailed above.

232

After the 24 h incubation period, the cells were homogenized and centrifuged at 10,000×g.

233

The supernatant was used to determine GPx activity in 96-well plates using a colorimetric

234

assay kit (BioVision, CA 95035, USA). GPx activity was measured by incubating the plates

235

at 25˚C for 15 min and reading fluorescence at 340 nm (Chen et al., 2014a). GPx activity is

236

expressed as mU/mg of protein. Units of GPx activity are expressed as the amount of enzyme

237

required to oxidize 1 nM NADPH per min.

M AN U

SC

RI PT

230

239

TE D

238

2.12. Flow cytometric evaluation of apoptosis Apoptotic cell death was analyzed by flow cytometry as described previously

241

(Kanthasamy et al., 2008). C6 cells (10×105) were seeded in 6-well plates and treated with

242

scopolamine, lactucopicrin, and/or galantamine as described above. Cells were then labeled

243

using an Annexin V-fluorescein Isothiocyanate (FITC) and propidium iodide (PI) staining kit

244

(Santa Cruz Biotech) as per the manufacturer’s protocol. Flow cytometry was performed by

245

Becton Dickenson FACs (Bacton Dickinson, San Francisco, CA). The excitation wavelength

246

for Annexin V-FITC dye was 488 nm and the emission wavelength was 530 nm. For PI

247

fluorescence, excitation was measured at 536 nm and emission was measured at 617 nm.

248

Apoptotic cell death is expressed as the percentage of apoptotic cells.

249

AC C

EP

240

ACCEPTED MANUSCRIPT 250

2.13. Statistical analysis All data are presented as the mean ± standard deviation of at least three independent

252

experiments. Statistical comparisons were performed using one-way analysis of variance

253

(ANOVA) followed by Tukey's post hoc test for multiple comparisons using GraphPad Prism

254

5.0 (GraphPad Software Inc., San Diego, CA, USA). P values less than 0.05 were considered

255

statistically significant and the degree of freedom (F) ratio was used to show the variance

256

between group means.

RI PT

251

258

SC

257

3. Results

260

M AN U

259

3.1. Lactucopicrin reduces scopolamine-induced toxicity in C6 glioma cells It was previously shown that scopolamine, a muscarinic acetylcholine receptor

262

(mAChR) antagonist, is cytotoxic to various neural cell types. To evaluate scopolamine-

263

induced toxicity in C6 glioma cells, cells were treated with various concentrations of

264

scopolamine (1, 3, 5, or 10 mM) for 1 h and then incubated with culture medium containing 2%

265

FBS for 24 h. Cell viability was examined with an MTT assay and the percentage of viable

266

cells was calculated relative to the vehicle-treated control. As shown in Figure 1A, cell

267

viability decreased (97.0±7.43, 77.9±4.70, 55.6±1.08, and 24.7±3.03%) with increasing

268

concentrations of scopolamine (1, 3, 5, and 10 mM, respectively), confirming that

269

scopolamine is cytotoxic to C6 glioma cells. ANOVA analysis for differences in

270

scopolamine-induced cytotoxicity between treatment groups revealed a P value of of 0.0001

271

with an F ratio of 76.31, which indicate statistically significant differences between group

272

means. Tukey's post-hoc test revealed that percent cell viability was significantly decreased

273

after cells were treated with 3 (p<0.05), 5 (p<0.01), and 10 mM (p<0.001) scopolamine,

274

compared to vehicle (Figure 1A). The cell viability at 3 mM scopolamine is considered to be

AC C

EP

TE D

261

ACCEPTED MANUSCRIPT 275

optimum to recover in 24 h period. Based on observation of cell toxicity at 5 mM

276

scopolamine, it showed high toxicity over 50% and recovering effect in 24 h period might be

277

unpredictable or low. Hence, we designed the further experiments by treatment of 3 mM

278

scopolamine. To examine the effects of lactucopicrin and galantamine, AChE inhibitors, on

280

scopolamine-induced toxicity, C6 cells were pre-treated with 3 mM scopolamine for 1 h and

281

then treated with 0.5, 1, or 2 µM lactucopicrin, or 2 µM galantamine as a positive control.

282

After 24 h incubation, cell viability was assessed with an MTT assay. Figure 1B shows that

283

the scopolamine-induced reduction in cell viability (70.5±2.46%) was significantly recovered

284

by 2 µM lactucopicrin (81.1±8.01, 82.7±7.92, and 88.6±3.74%), and by 2 µM galantamine

285

(97.5±9.15%). Statistical analysis for scopolamine-induced cytotoxicity showed an overall P

286

value for differences between the six groups (control, scopolamine-, lactucopicrin-, and

287

galantamine-treated groups) of 0.0009 with an F ratio of 9.063, which indicates statistically

288

significant differences between group means. Tukey's post-hoc analysis showed that percent

289

cell viability was significantly decreased after scopolamine (3 mM) treatment (p = 0.01) in

290

comparison with vehicle-treated control cells, whereas cell viability increased following

291

treatment with lactucopicrin (0.5, 1, or 2 µM) dose-dependently (2 µM, p<0.05) compared to

292

scopolamine treatment alone. These results suggest that lactucopicrin and galantamine are

293

protective against scopolamine-induced cytotoxicity in C6 glioma cells.

SC

M AN U

TE D

EP

AC C

294

RI PT

279

Scopolamine-induced cytotoxicity may be mediated by apoptotic cell death.

295

Apoptotic cell death was analyzed by flow cytometry after treating cells with scopolamine,

296

lactucopicrin, and/or galantamine as described above and labeling them with Annexin V-

297

FITC and PI. Quantification of apoptosis was based on the number of early (Annexin V) and

298

late (Annexin V+PI) apoptotic cells (Figure 1C, lower right and upper right quadrant,

299

respectively). Percent apoptosis is plotted as a bar graph (Figure 1D). Flow cytometry

ACCEPTED MANUSCRIPT analysis showed that scopolamine treatment increased apoptotic cell death by 10.77±0.67%

301

in comparison with vehicle treatment. Apoptotic events were markedly reduced by treatment

302

with lactucopicrin (0.5, 1, and 2 µM, by 5.44±1.23, 1.78±1.12, and 2.35±0.43%, respectively).

303

Galantamine (2 µM) also reduced apoptotic cell death, by 4.16±0.31%, but was not as

304

effective as lactucopicrin. The summed P value for differences between the number of

305

apoptotic events in the six groups was 0.0001 with an F ratio of 45.37. Tukey's post-hoc test

306

revealed significantly increased apoptotic cell death (p<0.001) in the scopolamine-treated

307

group compared with the vehicle-treated control group. Lactucopicrin treatment greatly

308

reduced apoptotic cell death in a concentration-dependent manner (0.5 µM, p<0.05; 1 µM,

309

p<0.01; and 2 µM, p=0.001) in comparison with scopolamine treatment alone.

M AN U

SC

RI PT

300

310

To confirm that lactucopicrin has an antiapoptotic effect, we also measured the levels of

312

cleaved and activated caspase 3 by Western blot (Figure 1E). The data clearly show that

313

scopolamine treatment increases cleavage/activation of caspase 3 (160.45±12.66%) and 0.5, 1,

314

and 2 µM lactucopicrin treatment abrogated scopolamine-induced caspase 3 activation to

315

85.19±7.02, 93.28±1.86, and 59.39±5.81% respectively. Galantamine (2 µM) also prevented

316

caspase 3 cleavage (51.7±2.88%) in a similar manner as lactucopicrin. The summed P value

317

for differences between the six groups was <0.0001 with an F ratio of 55.35, demonstrating

318

differences in expression of cleaved/activated caspase 3 between the control group and

319

experimental groups. Tukey's post-hoc test showed a marked increase in cleaved/activated

320

caspase 3 in scopolamine-treated cells (p<0.01) in comparison with vehicle-treated cells. The

321

scopolamine pre-treated lactucopicrin-treated group showed a marked reduction in cleaved/

322

activated caspase 3 (0.5 µM, p<0.01; 1 µM, p<0.01; and 2 µM, p<0.001) when compared

323

with the scopolamine-treated group. Taken together, the results suggest that lactucopicrin and

AC C

EP

TE D

311

ACCEPTED MANUSCRIPT 324

galantamine protect C6 glioma cells from scopolamine-induced cytotoxicity by reducing

325

apoptotic cell death.

326

3.2. Lactucopicrin ameliorates scopolamine-induced toxicity by reducing ROS

328

accumulation in C6 cells

RI PT

327

To determine whether lactucopicrin exerts its protective effect on scopolamine-

330

induced cytotoxicity by reducing ROS accumulation, cells were incubated with 3 mM

331

scopolamine for 1 h and then treated with various concentrations (0.5, 1, and 2 µM) of

332

lactucopicrin or 2 µM galantamine as a positive control for 24 h. The cells were then treated

333

with 30 µM DCFH-DA for 30 min and cellular ROS levels were measured by fluorescence

334

microscopy. The results showed that scopolamine treatment increased ROS levels to

335

177.5±12.31% of levels in the vehicle-treated control group (Figures 2A & 2B). Treatment

336

of scopolamine-pre-treated cells with lactucopicrin reduced DCF fluorescence intensity in a

337

dose-dependent manner (92.31±16.2, 75.6±8.98, and 70.5±11.34% for 0.5, 1, and 2 µM

338

lactucopicrin, respectively). Galantamine (2 µM) also reduced ROS levels (75.7±14.36%),

339

similarly to lactucopicrin. ROS levels were significantly different between the control and

340

treatment groups (summed P value = 0.0009, F ratio = 21.68). Tukey's post-hoc test showed a

341

significant increase in ROS levels (p<0.01) in the scopolamine-treated group compared to the

342

vehicle-treated control group. Scopolamine pre-treated, lactucopicrin-treated cells had

343

significantly reduced ROS levels (0.5 µM, p<0.01; 1 µM, p<0.01; and 2 µM, p<0.001) as

344

compared with scopolamine pre-treated cells. The results suggest that lactucopicrin protects

345

cells from scopolamine-induced oxidative stress by reducing ROS accumulation.

AC C

EP

TE D

M AN U

SC

329

346 347

3.3. Lactucopicrin reduces scopolamine-induced ∆ψm and cytochrome C release in C6

348

cells

ACCEPTED MANUSCRIPT 349

It has been suggested that scopolamine induces ROS accumulation upstream of

350

mitochondrial dysfunction and apoptosis (Choi et al., 2012; Guan et al., 2009). Thus, we

351

further

352

mitochondrial dysfunction and apoptosis. To examine the effect of scopolamine on ∆ψm loss

353

in C6 cells, cells were seeded on sterilized coverslips in 6-well plates and pre-treated with 3

354

mM scopolamine for 1 h. After further incubation with three concentrations (0.5, 1, and 2 µM)

355

of lactucopicrin or 2 µM galantamine as a positive control for 24 h, cells were fixed and

356

treated with 50 nM rhodamine 123 for 30 min, and fluorescence intensity was observed with

357

a confocal laser scanning microscope. The results showed that ∆ψm dramatically decreased

358

following 24 h treatment with scopolamine in C6 cells, and treatment with lactucopicrin or

359

galantamine significantly reversed the scopolamine-induced ∆ψm loss (Figure 3A).

whether scopolamine-induced

cytotoxicity is

mediated

through

M AN U

SC

RI PT

examined

It has been reported that oxidative stress and pro-apoptotic proteins enhance

361

mitochondrial dysfunction via the release of cytochrome C (Hou et al., 2015). To investigate

362

the protective role of lactucopicrin in scopolamine-induced mitochondrial dysfunction and

363

cytotoxicity, its effect on cytochrome C release was examined. Figure 3B shows that

364

scopolamine treatment reduced the level of mitochondrial cytochrome C to 77.9±9.88% and

365

increased that of cytosolic cytochrome C to 231.8±14.16%, which is indicative of

366

cytochrome C release. Upon treatment with lactucopicrin (0.5, 1, and 2 µM), there was a

367

reversal of cytochrome C secretion (Figure 3B), as evidenced by alterations in the level of

368

mitochondrial cytochrome C (101.27±11.92, 118.27±2.19, and 143.68±8.53%, respectively)

369

and cytosolic cytochrome C (87.24±10.27, 130.30±2.42, and 105.10±6.69%, respectively).

370

Galantamine (2 µM) also reversed cytochrome C release to a similar extent as 2 µM

371

lactucopicrin (132.0±2.34% for cytosolic cytochrome C and 157.1±2.79% for mitochondrial

372

cytochrome C). The ratios of mitochondrial to cytosolic cytochrome C in lactucopicrin-

373

treated cells were quite similar to that in the vehicle-treated control group (Figure 3C). The

AC C

EP

TE D

360

ACCEPTED MANUSCRIPT ratio of mitochondrial to cytosolic cytochrome C was significantly different between groups

375

(summed P value = 0.0242, F ratio = 6.07). Tukey's post-hoc test revealed that the

376

mitochondrial to cytosolic cytochrome C ratio was significantly decreased in the

377

scopolamine-treated group (p<0.05) compared with the vehicle-treated control group. 2 µM

378

lactucopicrin significantly increased the level of mitochondrial cytochrome C (p<0.05) in

379

comparison with scopolamine treatment alone. Taken together, these results suggest that

380

lactucopicrin and galantamine protect cells from scopolamine-induced cytotoxicity by

381

reducing ∆ψm reduction and cytochrome C release in C6 cells.

SC

RI PT

374

382

3.4. Lactucopicrin increases the levels of mAChR, p-AKT, and BcL-2 and reduces the

384

level of Bax, thereby reducing apoptosis

M AN U

383

Scopolamine may reduce acetylcholine-dependent signal transduction by acting as an

386

antagonist of mAChR, whereas lactucopicrin and galantamine may enhance it by increasing

387

the level of acetylcholine. Therefore, we examined the effects of these drugs on mAChR

388

expression and subsequent activation of signaling proteins including AKT in C6 cells.

389

mAChR protein levels were measured by Western blot after cells were pre-treated with 3 mM

390

scopolamine for 1 h and then treated with lactucopicrin or galantamine for 24 h. The results

391

showed that scopolamine pre-treatment alone did not significantly alter the expression of

392

mAChR (78.1±12.23% as compared to the vehicle-treated control group). Treatment with

393

either lactucopicrin (0.5, 1, and 2 µM) or galantamine (2 µM) increased mAChR levels to

394

114.2±22.42, 128.1±17.67, and 171.1±24.03%, or 114.8±11.5%, respectively (Figure 4)

395

(summed P value = 0.0335, F ratio = 5.271). Tukey's post-hoc test showed that the

396

scopolamine-pre-treated lactucopicrin (2 µM) –treated group had a significantly greater

397

mAChR level as compared with the scopolamine-treated group (p<0.05).

AC C

EP

TE D

385

ACCEPTED MANUSCRIPT The AKT pathway is a well-known signal transduction pathway that promotes cell

399

survival, growth, proliferation, migration, and angiogenesis. AKT is activated by

400

phosphorylation by phosphoinositide-dependent kinases, acts as a serine/threonine kinase,

401

and influences many factors involved in apoptosis (Nicholson and Anderson, 2002).

402

Therefore, we examined the levels of total and phosphorylated/activated AKT (p-AKT) by

403

Western blot. Figure 4 shows that scopolamine decreased the level of p-AKT (93.9±7.85%),

404

which was reversed by treatment with lactucopicrin or galantamine. The p-AKT level was

405

increased by treatment with lactucopicrin (0.5, 1, and 2 µM) in a dose-dependent manner

406

(104.9±6.66, 111.2±7.3, and 160.3±8.95%, respectively). Galantamine (2 µM) also increased

407

the p-AKT level (118.2±1.16%) when compared with that of the scopolamine-treated group

408

(993.9%). The difference between the groups was statistically significant (summed P value =

409

0.0551, F ratio = 4.19). Tukey's post-hoc test showed a significant decrease in pAKT and

410

AKT expression levels in the scopolamine-treated group as compared with the vehicle-treated

411

control group (p<0.05). These results suggest that lactucopicrin and galantamine induce

412

mAChR expression and activate mAChR-dependent signal transduction, leading to cell

413

growth and survival, as evidenced by increases in the level of p-AKT in C6 cells.

TE D

M AN U

SC

RI PT

398

We attempted to identify how lactucopicrin induces these alterations in scopolamine-

415

induced ROS production, mitochondrial dysfunction, and cytochrome C release. The

416

expressional regulation of the pro-apoptotic protein Bax and the antiapoptotic protein BcL-2

417

and their translocation to the mitochondria may be closely related to cytotoxicity. A previous

418

study demonstrated that scopolamine modulates the levels of apoptotic proteins (Moosavi et

419

al., 2012). Therefore, we assessed the levels of Bax and BcL-2 by Western blot analysis. The

420

results showed that scopolamine treatment decreases the level of BcL-2 (69.2±5.8%) and

421

increases that of Bax (141.6±13.56%) compared with vehicle treatment (Figure 4).

AC C

EP

414

ACCEPTED MANUSCRIPT As figure 4 shows, the alterations in BcL-2 and Bax were reversed by treatment with

423

lactucopicrin or galantamine. Treatment with lactucopicrin (0.5, 1, and 2 µM) significantly

424

increased the level of BcL-2 (124.8±6.33, 139.5±2.61, and 200.4±4.83%, respectively), as

425

compared with scopolamine treatment alone. Galantamine (2 µM) also increased the level of

426

BcL-2, to 178.0±9.99%. Treatment with lactucopicrin (0.5, 1, and 2 µM) significantly

427

decreased the level of Bax (133.5±13.94, 106.8±9.24, and 91.4±5.45%, respectively), when

428

compared with scopolamine treatment alone. Galantamine (2 µM) also reduced the level of

429

Bax, to 100.9±11.01%. The differences in BcL-2 and Bax levels between the six groups were

430

significant (summed P value = 0.0006, F ratio = 24.72). Tukey's post-hoc test revealed a

431

significant reduction of the BcL-2/Bax ratio in the scopolamine-treated group (p<0.05) as

432

compared with the vehicle-treated group. Scopolamine pre-treated, lactucopicrin-treated cells

433

had a significantly increased ratio of BcL-2/Bax (0.5 µM, p<0.05; 1 µM, p<0.01; and 2 µM,

434

p=0.001) in comparison with scopolamine-treated cells. These results suggest that

435

lactucopicrin and galantamine exert their functions through increases in mAChR expression

436

levels, and exert cytoprotective effects by inducing the antiapoptotic proteins BcL-2 and

437

AKT and by reducing levels of the pro-apoptotic protein Bax as well as cytochrome C release

438

in C6 cells.

EP

AC C

439

TE D

M AN U

SC

RI PT

422

440

3.5. Lactucopicrin increases the expression/activation of antioxidant enzymes via the

441

NRF2 pathway

442

Scopolamine-induced ROS generation and oxidative stress may be mediated by

443

antioxidant enzymes, such as SOD, catalase, and GPx. Therefore, we investigated whether

444

lactucopicrin affected the activities of these antioxidant enzymes. SOD activity decreased to

445

68.1±1.6% in scopolamine-treated cells, compared to vehicle-treated control cells

446

(83.8±1.67%). While lactucopicrin treatment (0.5, 1, and 2 µM) increased SOD activity in a

ACCEPTED MANUSCRIPT dose-dependent manner (68±0.84, 71±0.77, and 75.8±1.05%, respectively), galantamine (2

448

µM) did not have any effect on SOD activity (66.4±0.64) (Figure 5A) (summed P value =

449

<0.0001 and F ratio = 63.7 between corresponding groups). Tukey's post-hoc test showed a

450

significant depletion in SOD levels in the scopolamine-treated group (p<0.05 compared with

451

the vehicle-treated control group). Treatment with 2 µM lactucopicrin significantly restored

452

the SOD level (p<0.01). Taken together, these results clearly show that lactucopicrin

453

functions as an activator of SOD, unlike galantamine.

RI PT

447

Scopolamine treatment significantly reduced GPx activity to 97.8±5.35%, compared

455

with vehicle-treated control cells (112.2±2.58%). Lactucopicrin treatment (0.5, 1, and 2 µM)

456

of scopolamine pre-treated cells enhanced GPx activity (108±2.97, 107.6±3.18, and

457

108.7±2.47%, respectively) compared to scopolamine pre-treatment alone (Figure 5B).

458

Galantamine treatment of scopolamine-pre-treated cells also increased GPx activity, to

459

103.2±0.23%. The difference between the six groups was statistically significant (summed P

460

value = 0.151, F ratio = 7.407). Tukey's post-hoc test revealed a significant decrease in GPx

461

activity in the scopolamine-treated group as compared with the vehicle-treated control group

462

(p<0.05). The scopolamine pre-treated lactucopicrin-treated group had significantly enhanced

463

GPx activity in comparison with the scopolamine pre-treated group (p<0.05).

EP

TE D

M AN U

SC

454

Scopolamine pre-treatment also reduced catalase activity, to 0.54±0.04% compared

465

with vehicle treatment (1.42±0.21%). Catalase activity was increased to 1.36±0.15, 1.59±0.17,

466

and 1.66±0.15% by lactucopicrin treatment (0.5, 1, and 2 µM, respectively) following

467

scopolamine pre-treatment (Figure 5C). Galantamine treatment (2 µM) also improved

468

catalase activity, to 1.37±0.13%. The differences in catalase activity were statistically

469

significant (summed P value = 0.0028, F ratio = 14.27 between the treatment groups and the

470

control group). Tukey's post-hoc test revealed a significant reduction in catalase activity in

471

scopolamine-treated control as compared with vehicle-treated control cells (p<0.01). The

AC C

464

ACCEPTED MANUSCRIPT scopolamine pre-treated lactucopicrin-treated group showed a significant increase in GPx

473

level (0.5 µM, p<0.05; 1 µM, p<0.01; and 2 µM, p<0.01) in comparison with the

474

scopolamine-treated control group. These results suggest that lactucopicrin treatment

475

prevents scopolamine-induced toxicity by recovering the activities of catalase and other

476

antioxidant enzymes and by suppressing oxidative stress. Galantamine treatment also

477

reversed the scopolamine-induced reduction in antioxidant enzyme activity, but not as

478

effectively as lactucopicrin.

RI PT

472

It is well established that NRF2 is a transcription factor that induces endogenous

480

defense pathways and antioxidant enzymes and has the capacity to protect mitochondria in

481

neurodegenerative disease states (Ni et al., 2014). To assess scopolamine-induced alteration

482

of NRF2 expression in C6 glioma cells, we treated the cells with various concentrations of

483

scopolamine (1, 3, 5, and 10 mM) for 1 h, then refreshed the culture medium with 2% FBS

484

for 24 h. The expression level of NRF2 was determined by Western blot and compared with

485

that in the vehicle-treated control. As shown in Figures 5D and E, NRF2 expression

486

significantly decreased (92.0±8.0, 91.6±8.46, 72.2±0.97, and 62.1±6.7%) with an increase in

487

scopolamine concentration (1, 3, 5, and 10 mM, respectively). Statistical analysis showed that

488

scopolamine-induced cytotoxicity suppressed NRF2 expression (summed P value for the five

489

groups = 0.0116, F ratio = 10.65). Tukey's post-hoc test revealed that the percentage of NRF2

490

expression was significantly less following treatment with 5 (p<0.05) and 10 mM (p<0.05)

491

scopolamine compared to vehicle. Because lactucopicrin was found to stimulate the

492

expression of antioxidant enzymes and bring about an associated improvement in

493

mitochondrial function, as evidenced by recovery from ∆ψm loss and cytochrome C release in

494

scopolamine-treated C6 cells, we examined whether lactucopicrin and scopolamine affect the

495

level of NRF2 in C6 cells. We analyzed NRF2 protein levels by Western blot (Figures 5F

496

and G). Eventhough, 3 mM scopolamine treatment was not as effective as 5 mM, we first

AC C

EP

TE D

M AN U

SC

479

ACCEPTED MANUSCRIPT started performing western blot using 3 mM scopolamine to observed the effect of

498

lactucopicrin and galantamine. The results revealed that treatment with scopolamine alone

499

reduced the level of NRF2 to 87.6±3.77%, and that treatment with lactucopicrin (0.5, 1, and 2

500

µM) after scopolamine pre-treatment significantly increased NRF2 protein expression

501

(90.8±0.5, 110.0±4.42, and 108.9±2.56%, respectively). Treatment of scopolamine pre-

502

treated cells with galantamine (2 µM) also enhanced NRF2 expression, to 120.5±2.39%,

503

compared with the scopolamine alone-treated group. The statistical analysis showed that

504

NRF2 expression was significantly different between the control and treatment groups

505

(summed P value = 0.0016, F ratio = 17.54). Tukey's post-hoc test showed a significant

506

increase in NRF2 expression in scopolamine pre-treated lactucopicrin-treated cells in a

507

concentration-dependent manner (1 µM, p<0.05; 2 µM, p=0.05) as compared to scopolamine

508

treated control cells.

M AN U

SC

RI PT

497

From the figures 5D and F, it is clear that treatment of 3 mM scopolamine did not

510

show a significant difference in expression of NRF2 compared with control group. To justify

511

amelioration of scopolamine-induced oxidative stress by lactucopicrin, we again performed

512

the NRF2 expression by western blot technique using 5 mM scopolamine treatment. The

513

result showed significant reduction in the level of NRF2 as 61.4±6.18% than control group,

514

and in lactucopicrin (0.5, 1, and 2 µM) treatment after scopolamine 5 mM pre-treatment

515

significantly

516

128.4±13.37%, respectively). Galantamine (2 µM) treatment with scopolamine 5 mM pre-

517

treated cells also enhanced NRF2 expression, to 135.7±21.07%, compared with the

518

scopolamine alone-treated group. Statistical analysis showed that NRF2 expression was

519

significantly different between the control and treatment groups (summed P value = 0.0163,

520

F ratio = 7.168). Tukey's post-hoc test showed a significant increase in NRF2 expression in

521

scopolamine pre-treated lactucopicrin-treated cells in a concentration-dependent manner (1

AC C

EP

TE D

509

increased

NRF2

protein

expression

(100.2±7.01,

128.4±20.58,

and

ACCEPTED MANUSCRIPT 522

µM, p<0.05; 2 µM, p=0.05) as compared to scopolamine treated control cells. These results

523

suggest that lactucopicrin and galantamine protect C6 cells from scopolamine-induced

524

toxicity via the NRF2 pathway.

525

3.6. Lactucopicrin reduces scopolamine-induced toxicity by increasing the secretion of

527

the neurotrophic factor NGF and neurite outgrowth.

RI PT

526

It was previously reported that lactucopicrin treatment induces the secretion of

529

neurotrophic factors in glioma cells. The present study, therefore, measured changes in NGF

530

expression following treatment with lactucopicrin and scopolamine in C6 glioma cells. To

531

estimate the level of soluble NGF, C6 cells were seeded in 24-well plates and treated with

532

lactucopicrin or galantamine as described previously. After 24 h of treatment, the supernatant

533

was collected and assayed for NGF using a β-NGF ELISA kit. As shown in Figure 6A,

534

treatment of scopolamine pre-treated cells with lactucopicrin (0.5, 1, and 2 µM) significantly

535

induced secretion of NGF (92.8±6.51, 103.6±2.12, and 89.9±5.57%, respectively) when

536

compared to scopolamine treatment alone (78.1±0.79%). Interestingly, NGF secretion was

537

further reduced, to 44.8±9.75%, by treatment with 2 µM galantamine. Statistical analysis

538

showed significant changes in NGF secretion between the control and experimental groups

539

(summed P value = 0.0003, F ratio = 31.95). Tukey's post-hoc test revealed a significant

540

decrease in NGF secretion in the scopolamine-treated control group compared with the

541

vehicle-treated control group (p<0.05). Scopolamine pre-treated cells treated with 1 µM

542

lactucopicrin exhibited an improvement in NGF levels (p<0.05) in comparison with the

543

scopolamine-treated control group. These results suggest that lactucopicrin exerts its

544

neuroprotective effect in part by promoting NGF secretion in scopolamine-treated C6 glioma

545

cells.

AC C

EP

TE D

M AN U

SC

528

ACCEPTED MANUSCRIPT Scopolamine was shown to have a potent negative impact on neurite outgrowth in

547

neuronal cells (Yamazaki et al., 2001). To examine the effect of lactucopicrin-induced NGF

548

secretion on neurite outgrowth, N2a neuroblastoma cells were treated with scopolamine and

549

lactucopicrin for 24 h, as was done to C6 glioma cells. Retinoic acid and galantamine were

550

used in this experiment as positive controls for neurite outgrowth. Automated images were

551

captured using an Essen IncuCyte, and the images were analyzed for neurite outgrowth. As

552

expected, retinoic acid and galantamine enhanced neurite outgrowth in N2a cells

553

(196.2±12.13% and 217.4±32.65%, respectively) (Figures 6B and C). Scopolamine

554

treatment alone significantly attenuated neurite outgrowth (71.8±1.17% compared with

555

vehicle treatment), whereas lactucopicrin (0.5, 1, and 2 µM) treatment of scopolamine-pre-

556

treated N2a cells significantly promoted neurite outgrowth in a dose-dependent manner

557

(222.7±36.16, 244.4±32.38, and 259.6±20.22%, respectively). Statistical analysis of neurite

558

outgrowth showed significance with (summed P value = 0.0006 and F ratio = 18.31 between

559

the control group and the corresponding treatment groups. Tukey's post-hoc demonstrated a

560

reduction in neurite outgrowth in the scopolamine-treated control group compared with the

561

vehicle-treated control group (p<0.05). Scopolamine pre-treated lactucopicrin treatment

562

significantly induced neurite outgrowth in a concentration-dependent manner with 0.5 µM

563

(p<0.05), 1 µM (p<0.01), and 2 µM (p=0.01) as compared with scopolamine treatment alone.

564

Neurite outgrowth induced by lactucopicrin was greater than that induced by galantamine and

565

retinoic acid. Taken together, these results show that lactucopicrin ameliorates scopolamine-

566

induced neurotoxicity.

AC C

EP

TE D

M AN U

SC

RI PT

546

567 568

4. Discussion

569

Neurodegenerative diseases, such as AD, and other dementias usually characterized

570

with decline in memory and cognitive function. It is associated with cholinergic neuronal cell

ACCEPTED MANUSCRIPT death in basal forebrain (Crapper and DeBoni, 1978; Ghezzi et al., 2013). Antimuscarinic

572

syndrome brought about by scopolamine is associated with impairments in cognitive function

573

and psychosis, which are also symptoms of neurodegeneration (Barak and Weiner, 2009).

574

Thus, scopolamine induced cognitive dysfunction was considered as important AD model

575

(Lee et al., 2014; Moosavi et al., 2012; Pandareesh and Anand, 2013). Using N2a mouse

576

neuroblastoma cell line for scopolamine AD model, we can assess the neurotoxicity and

577

neuroprotection (Laufs et al., 2004). Only neuron-oriented research can't overcome the

578

Alzheimer's disease. Also, glia cells may be a key elements of Alzheimer´s disease. So, it has

579

been suggested that glia cells play an important role in the proper accomplishment of

580

cognitive tasks (Hilgetag and Barbas, 2009). Additionally, astrocytes surrounding neurons are

581

involved in supplying nutrients and oxygen, destroying pathogens, and eliminating dead

582

neurons, thereby playing supportive and protective roles in the CNS (Guérout et al., 2014).

583

So, it seems that c6 glioma cells based research is worthwhile in the neurodegenerative field.

584

Hence, scopolamine-mediated toxicity in glioma and neuronal cells is an effective, beneficial,

585

and justifiable model of AD and correlates with the effects of scopolamine in other systems.

TE D

M AN U

SC

RI PT

571

Age related diseases and AD pathogenesis ultimately causes ROS accumulation in

587

neuronal cells evidenced with cytochrome C release, ∆ψm loss, caspase activation, and DNA

588

damages (Rönnbäck et al., 2015). Moreover, AD patients present with reductions in

589

antioxidant enzyme levels and increases in oxidative stress in the temporal cortex (Marcus et

590

al., 1998). Okadaic acid induced ROS oxidative stress with suppression in the antioxidative

591

enzyme and ∆ψm in mouse hippocampal neurons were attenuated by quercetin reported as

592

potential therapeutic for AD (Jiang et al., 2016). Therefore, in this study, we utilized

593

scopolamine-pre-treated C6 glioma cells as a AD model and N2a neuroblastoma cells to

594

investigate neurite outgrowth.

AC C

EP

586

ACCEPTED MANUSCRIPT An abundance of evidence supports the use of scopolamine to produce a model of

596

neurodegenerative disease (Konar et al., 2011). In the present study, we observed

597

scopolamine-induced cytotoxic effects in C6 glioma cells, as evidenced by reduced cell

598

viability (Figure 1A), via induction of apoptosis (Figure 1C and D). Scopolamine

599

commenced apoptosis by activation of caspase 3 directed apoptotic cell death (Figures 1E

600

and F) with alteration in pro-apoptotic proteins and inhibition of antiapoptotic proteins

601

(Figures 4A). Hydrogen peroxide induced oxidative stress and free radical in human neuroblastoma

602

cell (SH-SY5Y). It also decreases ratio of BcL2/Bax and activation of caspase 3 and 9 mediated

603

apoptotic cell death (Hu et al., 2015). Further, scopolamine treatment increased ROS

604

accumulation (Figure 2A and B). Mitochondria, a coordinator for energy metabolism homeostasis

605

and apoptosis contributes to many cellular functions of nervous system (Harper et al., 2004; Shulman

606

et al., 2004). AD pathogenesis with mitochondrial dysfunction (Schmitt et al., 2012). due to

607

increased consumption of ATP while in disruption of mitochondrial integrity enhance the loss

608

of ∆ψm (Figure 3A) and cytochrome C release (Figures 3B and C). It further generates

609

ROS accumulation and induces apoptosis. Scopolamine being a tropane alkaloid with non-

610

selective muscarinic antagonist inhibits mAChR with hindrance in cholinergic activity in the

611

central nervous system (Lee et al., 2012). These studies again supported that scopolamine (3

612

mM) reduced mAChR expression (Figure 4A) in C6 glioma cells. Additionally, oxidative

613

stress believed to promote disturbance in antioxidant enzyme system imbalance (Grimm et al.,

614

2016), thus observed for scopolamine suppressed antioxidant enzyme levels (SOD, GPx, and

615

catalase) (Figures 5A, B and C) and the levels of the master transcription factor NRF2

616

(Figures 5D, E, F, G, H and I), which corresponded to reduced NGF secretion (Figure 6A)

617

and changes in neurite outgrowth in N2a cells (Figures 6B and C).

AC C

EP

TE D

M AN U

SC

RI PT

595

618

Several previous studies investigated the protective effects of plant extracts and

619

phytochemicals against scopolamine-induced cytotoxicity in neurodegenerative disease

ACCEPTED MANUSCRIPT models (Kim et al., 2014b). Here, we studied lactucopicrin, a type of sesquiterpene lactone

621

isolated from Lactuca virosa and Cichorium intybus. Lactucopicrin has analgesic, sedative

622

(Wesołowska et al., 2006), and acetylcholinesterase inhibitory activity (Rollinger et al., 2005).

623

Furthermore, galantamine, a drug currently on the market for the treatment of AD (Kim et al.,

624

2014b), was used as a positive control to assess the efficacy of lactucopicrin on scopolamine-

625

mediated toxicity in glioma and neuronal cells. Our data showed that lactucopicrin, like

626

galantamine, effectively promotes cell proliferation in a scopolamine-induced model of

627

neurodegeneration by significantly increasing C6 cell viability (Figure 1B) and reducing

628

ROS accumulation (Figures 2A and B). An oxidative stress and apoptotic cell death by

629

H2O2 and okadaic acid were ameliorated by kukoamine B and quercetin with induction in

630

restoring ∆ψm in SH-SY5Y and hippocampal neuronal cells (Hu et al., 2015; Jiang et al., 2016).

631

Scopolamine blockades in muscarinic M1 synaptic transmission, it disrupts

632

hippocampal cholinergic signalling pathway (Lee et al., 2012). To reveal the molecular

633

mechanisms underlying the effects of lactucopicrin on scopolamine-induced toxicity, we

634

further examined the levels of the acetylcholine receptor mAChR, a signaling protein

635

(phosphorylated AKT), and antiapoptotic and proapoptotic proteins (BcL-2 and Bax,

636

respectively) (Figures 4A and B). Our results clearly showed that lactucopicrin enhances the

637

levels of mAChR and p-Akt in a manner similar to galantamine. This is consistent with

638

reports that agmatine and arabinoxylan protect adult rat hippocampal cells from scopolamine-

639

induced toxicity by activating AKT and ERK (Kim et al., 2014a; Moosavi et al., 2012).

640

Allantoin also enhances memory by increasing hippocampal neuronal cell proliferation via

641

activation of the PI3K/AKT/GSK-3β signaling pathway following scopolamine treatment

642

(Ahn et al., 2014). Our observations and those of related reports suggest that lactucopicrin

643

upregulates the acetylcholine receptor-dependent PI3K/AKT signaling pathway, which may

644

be responsible for cell survival and neuroprotection in this disease model.

AC C

EP

TE D

M AN U

SC

RI PT

620

ACCEPTED MANUSCRIPT In an in vitro neurodegenerative disease model, ladostigil, rasagiline, and other

646

propargylamines were found to have neuroprotective effects that are attributable to the close

647

association of proteins involved in apoptosis, such as BcL-2, BcL-xl, Bax, and Bad, with

648

suppression of mitochondrial dysfunction in neurodegenerative disease states (Youdim et al.,

649

2005). Moreover, plant extracts and phytochemicals play a major role in neuroprotection and

650

neurogenesis owing to their antiapoptotic effects in cognitive dysfunction-related disorders

651

(Chen et al., 2014b; Jahanshahi et al., 2013; Jang et al., 2010; Kim and Ryu, 2008). In the

652

present study, we observed that lactucopicrin increases the level of BcL-2 while decreasing

653

that of Bax (Figures 4A and B). Alterations in the levels of BcL-2 and Bax by lactucopicrin

654

may explain its ability to reduce apoptotic cell death. Lactucopicrin prevents apoptotic cell

655

death via recovery of ∆ψm loss and mitochondrial cytochrome C release (Figures 3A, B and

656

C). Correspondingly, apoptotic cell death induced by scopolamine was ameliorated by

657

treatment with lactucopicrin and galantamine.

M AN U

SC

RI PT

645

Kukoamine B and quercetin reported as potent inhibitor of okadaic acid and H2O2

659

induced oxidative stress via inducing SOD, GPx, and catalase (Hu et al., 2015; Jiang et al.,

660

2016). Scopolamine induced a marked reduction in NRF2 expression in a dose-dependent

661

manner (Figures 5D and 5E), along with alterations in antioxidant enzyme levels.

662

Furthermore, lactucopicrin against scopolamine (3 or 5 mM) enhanced the expression of

663

antioxidant enzymes (Figures 5A, B, and C) and increased the expression of NRF2 (Figure

664

5F and H) in a similar pattern as galantamine. As lactucopicrin enhanced the activities of

665

antioxidant enzymes, such as SOD, GPx, and catalase, and that of the master transcription

666

factor NRF2 (Figures 5A, B, C, F and H), it may reduce ROS accumulation and oxidative

667

stress-induced damage via increased NRF2-dependent expression of antioxidant enzymes.

668

Overexpression of NRF2 and neuroprotection mediated by enhancements in antioxidant

669

enzymes have also been shown in other neurodegenerative disease models. Similar to

AC C

EP

TE D

658

ACCEPTED MANUSCRIPT 670

lactucopicrin, aloe-emodin (Tao et al., 2014) and ladostigil (Weinreb et al., 2012) were

671

shown to reverse scopolamine-induced toxicity by reducing the levels of ROS via induction

672

of antioxidant enzymes. Phenolic derivatives of Dioscorea sp. (Woo et al., 2014) and the bark of Eucommia sp.

674

(Kwon et al., 2013) showed neuroprotective activity by increasing BDNF levels and NGF

675

secretion in glioma cells and significantly enhancing neurite outgrowth in neuroblastoma

676

cells. Lactucopicrin also increased glioma cell NGF secretion (Figure 6A) and N2a cell

677

neurite outgrowth following scopolamine treatment (Figure 6B). Galantamine was not as

678

effective as lactucopicrin in enhancing NGF secretion and neurite outgrowth. A previous

679

study on ladostigil showed that it has similar protective effects against scopolamine-mediated

680

toxicity by strengthening neurotrophic support in the rat brain (Weinreb et al., 2012).

681

Scopolamine-induced cognitive deficits were improved by PF-04447943, a synthetic

682

compound, by enhancement of synaptic activity in rodent hippocampal neuronal cultures as

683

measured by neurite outgrowth length and number of synapses (Hutson et al., 2011). In light

684

of these beneficial effects, lactucopicrin should be considered as a neuroprotective agent

685

against scopolamine-induced AD model.

TE D

M AN U

SC

RI PT

673

Given our findings, we propose a molecular mechanism for the novel cytoprotective

687

effect of lactucopicrin. Scopolamine reduces the effect of the mAchR acetylcholine receptor,

688

resulting in ROS generation and subsequent mitochondrial dysfunction. These alterations

689

may be responsible for the activation of the mitochondrial apoptosis pathway and cytotoxicity.

690

Lactucopicrin enhances the level of acetylcholine and the expression of mAChR via

691

inhibition of AChE. The AKT signaling pathway may play important roles in cell

692

proliferation, expression of the neurotrophic factor NGF, and neurite outgrowth, and have

693

various antiapoptotic functions including modulation of proapoptotic Bax and antiapoptotic

694

BcL-2. These factors are closely related to alteration in ROS generation and mitochondrial

AC C

EP

686

ACCEPTED MANUSCRIPT dysfunction. Lactucopicrin also induce antioxidant enzymes, presumably via activation of the

696

master transcription factor NRF2, which might rescue cells from ROS-mediated cytotoxicity.

697

These neuroprotective actions of lactucopicrin are indicative of its potential in the treatment

698

of neurodegenerative diseases. Further knockdown and in vivo studies are needed to confirm

699

the proposed mechanisms of action of lactucopicrin.

700 701

Authors’ contributions

RI PT

695

RV and SYK designed the research and wrote the manuscript. RV performed the

703

experiments and interpreted the data. RV and LS analyzed the data. EJY contributed through

704

valuable suggestions for experiments and for refinement of the manuscript. All authors

705

critically evaluated the manuscript. All authors have read and approved the final version of

706

the manuscript.

M AN U

SC

702

707

Acknowledgements

709

This research was supported by the Bio & Medical Technology Development Program of the

710

NRF funded by the Korean government, MSIP (NRF-2014M3A9B6069338).

EP

TE D

708

711

Conflicts of Interest

713

The authors declare no conflicts of interest.

714

AC C

712

715

References

716

Aebi, H., 1984. Catalase in vitro. 121-125. Methods in enzymology 105.

717

Ahn, Y.J., Park, S.J., Woo, H., Lee, H.E., Kim, H.J., Kwon, G., Gao, Q., Jang, D.S., Ryu,

718

J.H., 2014. Effects of allantoin on cognitive function and hippocampal neurogenesis. Food

719

and Chemical Toxicology 64, 210-216.

ACCEPTED MANUSCRIPT Bajo, R., Pusil, S., López, M., Canuet, L., Pereda, E., Osipova, D., Maestú, F., Pekkonen, E.,

721

2015. Scopolamine effects on functional brain connectivity: a pharmacological model of

722

Alzheimer’s disease. Scientific reports 5; 9748.

723

Barak, S., Weiner, I., 2009. Towards an animal model of an antipsychotic drug-resistant

724

cognitive impairment in schizophrenia: scopolamine induces abnormally persistent latent

725

inhibition, which can be reversed by cognitive enhancers but not by antipsychotic drugs. The

726

International Journal of Neuropsychopharmacology 12, 227-241.

727

Blennow, K., Mattsson, N., Schöll, M., Hansson, O., Zetterberg, H., 2015. Amyloid

728

biomarkers in Alzheimer's disease. Trends in Pharmacological Sciences.

729

Brookmeyer, R., Johnson, E., Ziegler-Graham, K., Arrighi, H.M., 2007. Forecasting the

730

global burden of Alzheimer’s disease. Alzheimer's & dementia 3, 186-191.

731

Chen, M.-H., Tsai, C.-F., Hsu, Y.-W., Lu, F.-J., 2014a. Epigallocatechin gallate eye drops

732

protect against ultraviolet B–induced corneal oxidative damage in mice. Molecular vision 20,

733

153.

734

Chen, W., Cheng, X., Chen, J., Yi, X., Nie, D., Sun, X., Qin, J., Tian, M., Jin, G., Zhang, X.,

735

2014b. Lycium barbarum polysaccharides prevent memory and neurogenesis impairments in

736

scopolamine-treated rats. PloS one 9, e88076.

737

Choi, J.H., Lee, J.Y., Choi, A.-Y., Hwang, K.-Y., Choe, W., Yoon, K.-S., Ha, J., Yeo, E.-J.,

738

Kang, I., 2012. Apicidin induces endoplasmic reticulum stress-and mitochondrial

739

dysfunction-associated apoptosis via phospholipase Cγ1-and Ca2+-dependent pathway in

740

mouse Neuro-2a neuroblastoma cells. Apoptosis 17, 1340-1358.

741

Crapper, D., DeBoni, U., 1978. Brain aging and Alzheimer's disease. The Canadian

742

Psychiatric Association Journal/La Revue de l'Association des psychiatres du Canada.

743

Dzamba, D., Harantova, L., Butenko, O., Anderova, M., 2016. Glial cells-the key elements of

744

Alzheimer´ s disease. Current Alzheimer Research.

AC C

EP

TE D

M AN U

SC

RI PT

720

ACCEPTED MANUSCRIPT Ghezzi, L., Scarpini, E., Galimberti, D., 2013. Disease-modifying drugs in Alzheimer’s

746

disease. Drug design, development and therapy 7, 1471.

747

Godyń, J., Jończyk, J., Panek, D., Malawska, B., 2016. Therapeutic strategies for Alzheimer's

748

disease in clinical trials. Pharmacological Reports 68, 127-138.

749

Grimm, A., Mensah-Nyagan, A.G., Eckert, A., 2016. Alzheimer, mitochondria and gender.

750

Neuroscience & Biobehavioral Reviews.

751

Guan, L., Han, B., Li, Z., Hua, F., Huang, F., Wei, W., Yang, Y., Xu, C., 2009. Sodium

752

selenite induces apoptosis by ROS-mediated endoplasmic reticulum stress and mitochondrial

753

dysfunction in human acute promyelocytic leukemia NB4 cells. Apoptosis 14, 218-225.

754

Guérout, N., Li, X., Barnabé-Heider, F., 2014. Cell fate control in the developing central

755

nervous system. Experimental cell research 321, 77-83.

756

Haerter, F., Eikermann, M., 2016. Reversing neuromuscular blockade: inhibitors of the

757

acetylcholinesterase versus the encapsulating agents sugammadex and calabadion. Expert

758

Opinion on Pharmacotherapy.

759

Harper, M.E., Bevilacqua, L., Hagopian, K., Weindruch, R., Ramsey, J., 2004. Ageing,

760

oxidative stress, and mitochondrial uncoupling. Acta physiologica Scandinavica 182, 321-

761

331.

762

Hasselmann, H., 2014. Scopolamine and depression: a role for muscarinic antagonism? CNS

763

& Neurological Disorders-Drug Targets (Formerly Current Drug Targets-CNS &

764

Neurological Disorders) 13, 673-683.

765

Hilgetag, C.C., Barbas, H., 2009. Are there ten times more glia than neurons in the brain?

766

Brain Structure and Function 213, 365-366.

767

Hou, X., Tong, Q., Wang, W., Xiong, W., Shi, C., Fang, J., 2015. Dihydromyricetin protects

768

endothelial cells from hydrogen peroxide-induced oxidative stress damage by regulating

769

mitochondrial pathways. Life Sciences.

AC C

EP

TE D

M AN U

SC

RI PT

745

ACCEPTED MANUSCRIPT Hu, X.-L., Niu, Y.-X., Zhang, Q., Tian, X., Gao, L.-Y., Guo, L.-P., Meng, W.-H., Zhao, Q.-

771

C., 2015. Neuroprotective effects of Kukoamine B against hydrogen peroxide-induced

772

apoptosis and potential mechanisms in SH-SY5Y cells. Environmental toxicology and

773

pharmacology 40, 230-240.

774

Hutson, P., Finger, E., Magliaro, B., Smith, S., Converso, A., Sanderson, P., Mullins, D.,

775

Hyde, L., Eschle, B., Turnbull, Z., 2011. The selective phosphodiesterase 9 (PDE9) inhibitor

776

PF-04447943 (6-[(3S, 4S)-4-methyl-1-(pyrimidin-2-ylmethyl) pyrrolidin-3-yl]-1-(tetrahydro-

777

2H-pyran-4-yl)-1, 5-dihydro-4H-pyrazolo [3, 4-d] pyrimidin-4-one) enhances synaptic

778

plasticity and cognitive function in rodents. Neuropharmacology 61, 665-676.

779

Jahanshahi, M., Nickmahzar, E., Babakordi, F., 2013. The effect of Ginkgo biloba extract on

780

scopolamine-induced apoptosis in the hippocampus of rats. Anatomical science international

781

88, 217-222.

782

Jang, J.H., Kim, C.Y., Lim, S.H., Yang, C.H., Song, K.S., Han, H.S., Lee, H.K., Lee, J., 2010.

783

Neuroprotective effects of Triticum aestivum L. against β‐Amyloid‐induced cell death and

784

memory impairments. Phytotherapy Research 24, 76-84.

785

Jiang, W., Luo, T., Li, S., Zhou, Y., Shen, X.-Y., He, F., Xu, J., Wang, H.-Q., 2016.

786

Quercetin Protects against Okadaic Acid-Induced Injury via MAPK and PI3K/Akt/GSK3β

787

Signaling Pathways in HT22 Hippocampal Neurons. PloS one 11, e0152371.

788

Kang, J.-M., Ju, H.-L., Sohn, W.-M., Na, B.-K., 2014. Characterization of biochemical

789

properties of a selenium-independent glutathione peroxidase of Cryptosporidium parvum.

790

Parasitology 141, 570-578.

791

Kang, T.H., Moon, E., Hong, B.N., Choi, S.Z., Son, M., Park, J.-H., Kim, S.Y., 2011.

792

Diosgenin from Dioscorea nipponica ameliorates diabetic neuropathy by inducing nerve

793

growth factor. Biological and Pharmaceutical Bulletin 34, 1493-1498.

AC C

EP

TE D

M AN U

SC

RI PT

770

ACCEPTED MANUSCRIPT Kanthasamy, A.G., Kitazawa, M., Yang, Y., Anantharam, V., Kanthasamy, A., 2008.

795

Environmental neurotoxin dieldrin induces apoptosis via caspase-3-dependent proteolytic

796

activation of protein kinase C delta (PKCdelta): Implications for neurodegeneration in

797

Parkinson's disease. Molecular brain 1, 12.

798

Kása, P., Rakonczay, Z., Gulya, K., 1997. The cholinergic system in Alzheimer's disease.

799

Progress in neurobiology 52, 511-535.

800

Kim, C.-Y., Lee, G.-Y., Park, G.H., Lee, J., Jang, J.-H., 2014a. Protective effect of

801

arabinoxylan against scopolamine-induced learning and memory impairment. Biomolecules

802

& therapeutics 22, 467.

803

Kim, D.-H., Ryu, J.-H., 2008. Differential effects of scopolamine on memory processes in the

804

object recognition test and the Morris water maze test in mice. Biomolecules and

805

Therapeutics 16, 173-178.

806

Kim, M.H., Kim, S., Yang, W.M., 2014b. Mechanisms of action of phytochemicals from

807

medicinal herbs in the treatment of Alzheimerʼs disease. Planta Med 80, 1249-1258.

808

Konar, A., Shah, N., Singh, R., Saxena, N., Kaul, S.C., Wadhwa, R., Thakur, M.K., 2011.

809

Protective role of Ashwagandha leaf extract and its component withanone on scopolamine-

810

induced changes in the brain and brain-derived cells. PloS one 6, e27265.

811

Kwon, S.-H., Ma, S.-X., Joo, H.-J., Lee, S.-Y., Jang, C.-G., 2013. Inhibitory effects of

812

Eucommia ulmoides Oliv. Bark on scopolamine-induced learning and memory deficits in

813

mice. Biomolecules and Therapeutics 21, 462-469.

814

Laufs, T.L., Wystub, S., Reuss, S., Burmester, T., Saaler-Reinhardt, S., Hankeln, T., 2004.

815

Neuron-specific expression of neuroglobin in mammals. Neuroscience letters 362, 83-86.

816

Lee, B., Sur, B., Shim, I., Lee, H., Hahm, D.-H., 2012. Phellodendron amurense and its major

817

alkaloid compound, berberine ameliorates scopolamine-induced neuronal impairment and

818

memory dysfunction in rats. The Korean Journal of Physiology & Pharmacology 16, 79-89.

AC C

EP

TE D

M AN U

SC

RI PT

794

ACCEPTED MANUSCRIPT Lee, J.-S., Kim, H.-G., Han, J.-M., Kim, D.-W., Yi, M.-H., Son, S.-W., Kim, Y.-A., Lee, J.-S.,

820

Choi, M.-K., Son, C.-G., 2014. Ethanol extract of Astragali Radix and Salviae Miltiorrhizae

821

Radix, Myelophil, exerts anti-amnesic effect in a mouse model of scopolamine-induced

822

memory deficits. Journal of ethnopharmacology 153, 782-792.

823

Lee, Y.J., Hoe, K.L., Maeng, P.J., 2007. Yeast cells lacking the CIT1-encoded mitochondrial

824

citrate synthase are hypersusceptible to heat-or aging-induced apoptosis. Molecular biology

825

of the cell 18, 3556-3567.

826

León,

827

multitarget‐directed ligands approach for the treatment of Alzheimer's disease. Medicinal

828

research reviews 33, 139-189.

829

Li, G., Kim, C., Kim, J., Yoon, H., Zhou, H., Kim, J., 2015. Common Pesticide,

830

Dichlorodiphenyltrichloroethane (DDT), Increases Amyloid-β Levels by Impairing the

831

Function of ABCA1 and IDE: Implication for Alzheimer's Disease. Journal of Alzheimer's

832

Disease.

833

Marcus, D.L., Thomas, C., Rodriguez, C., Simberkoff, K., Tsai, J.S., Strafaci, J.A., Freedman,

834

M.L., 1998. Increased peroxidation and reduced antioxidant enzyme activity in Alzheimer's

835

disease. Experimental neurology 150, 40-44.

836

Moon, E., Lee, S.O., Kang, T.H., Kim, H.J., Choi, S.Z., Son, M.-W., Kim, S.Y., 2014.

837

Dioscorea extract (DA-9801) modulates markers of peripheral neuropathy in type 2 diabetic

838

db/db mice. Biomolecules & therapeutics 22, 445-452.

839

Moosavi, M., Khales, G.Y., Abbasi, L., Zarifkar, A., Rastegar, K., 2012. Agmatine protects

840

against scopolamine-induced water maze performance impairment and hippocampal ERK

841

and Akt inactivation. Neuropharmacology 62, 2018-2023.

J.,

2013.

Recent

SC

Marco‐Contelles,

advances

in

the

M AN U

A.G.,

TE D

Garcia,

AC C

EP

R.,

RI PT

819

ACCEPTED MANUSCRIPT Murray, A.P., Faraoni, M.B., Castro, M.J., Alza, N.P., Cavallaro, V., 2013. Natural AChE

843

inhibitors from plants and their contribution to Alzheimer’s disease therapy. Current

844

neuropharmacology 11, 388.

845

Ni, N., Liu, Q., Ren, H., Wu, D., Luo, C., Li, P., Wan, J.-B., Su, H., 2014. Ginsenoside Rb1

846

protects rat neural progenitor cells against oxidative injury. Molecules 19, 3012-3024.

847

Nicholson, K.M., Anderson, N.G., 2002. The protein kinase B/Akt signalling pathway in

848

human malignancy. Cellular signalling 14, 381-395.

849

Ogawa, S., 2014. Nutritional management of older adults with cognitive decline and

850

dementia. Geriatrics & gerontology international 14, 17-22.

851

Pandareesh, M., Anand, T., 2013. Neuromodulatory propensity of Bacopa monniera against

852

scopolamine-induced cytotoxicity in PC12 cells via down-regulation of AChE and up-

853

regulation of BDNF and muscarnic-1 receptor expression. Cellular and molecular

854

neurobiology 33, 875-884.

855

Rollinger, J., Mock, P., Zidorn, C., Ellmerer, E., Langer, T., Stuppner, H., 2005. Application

856

of the in combo screening approach for the discovery of non-alkaloid acetylcholinesterase

857

inhibitors from Cichorium intybus. Current drug discovery technologies 2, 185-193.

858

Rönnbäck, A., Pavlov, P., Mansory, M., Gonze, P., Marlière, N., Winblad, B., Graff, C.,

859

Behbahani, H., 2015. Mitochondrial dysfunction in a transgenic mouse model expressing

860

human amyloid precursor protein (APP) with the Arctic mutation. Journal of neurochemistry.

861

Schmitt, K., Grimm, A., Kazmierczak, A., Strosznajder, J.B., Götz, J., Eckert, A., 2012.

862

Insights into Mitochondrial Dysfunction: Aging, Amyloid-β, and Tau–A Deleterious Trio.

863

Antioxidants & redox signaling 16, 1456-1466.

864

Shulman, R.G., Rothman, D.L., Behar, K.L., Hyder, F., 2004. Energetic basis of brain

865

activity: implications for neuroimaging. Trends in neurosciences 27, 489-495.

AC C

EP

TE D

M AN U

SC

RI PT

842

ACCEPTED MANUSCRIPT Tao, L., Xie, J., Wang, Y., Wang, S., Wu, S., Wang, Q., Ding, H., 2014. Protective effects of

867

aloe-emodin on scopolamine-induced memory impairment in mice and H 2 O 2-induced

868

cytotoxicity in PC12 cells. Bioorganic & medicinal chemistry letters 24, 5385-5389.

869

Tohgi, H., Abe, T., Kimura, M., Saheki, M., Takahashi, S., 1996. Cerebrospinal fluid

870

acetylcholine and choline in vascular dementia of Binswanger and multiple small infarct

871

types as compared with Alzheimer-type dementia. Journal of neural transmission 103, 1211-

872

1220.

873

Wang, P., Wingnang Leung, A., Xu, C., 2011. Low-intensity ultrasound-induced cellular

874

destruction and autophagy of nasopharyngeal carcinoma cells. Experimental and therapeutic

875

medicine 2, 849-852.

876

Weinreb, O., Amit, T., Bar-Am, O., BH Youdim, M., 2012. Ladostigil: a novel multimodal

877

neuroprotective drug with cholinesterase and brain-selective monoamine oxidase inhibitory

878

activities for Alzheimer's disease treatment. Current drug targets 13, 483-494.

879

Wesołowska, A., Nikiforuk, A., Michalska, K., Kisiel, W., Chojnacka-Wójcik, E., 2006.

880

Analgesic and sedative activities of lactucin and some lactucin-like guaianolides in mice.

881

Journal of ethnopharmacology 107, 254-258.

882

Woo, K.W., Kwon, O.W., Kim, S.Y., Choi, S.Z., Son, M.W., Kim, K.H., Lee, K.R., 2014.

883

Phenolic derivatives from the rhizomes of Dioscorea nipponica and their anti-

884

neuroinflammatory and neuroprotective activities. Journal of ethnopharmacology 155, 1164-

885

1170.

886

Xiong, W., Garfinkel, A.E.M., Li, Y., Benowitz, L.I., Cepko, C.L., 2015. NRF2 promotes

887

neuronal survival in neurodegeneration and acute nerve damage. The Journal of clinical

888

investigation 125, 0-0.

889

Yamazaki, M., Hirakura, K., Miyaichi, Y., IMAKURA, K., KITA, M., CHIBA, K., MOHRI,

890

T., 2001. Effect of polyacetylenes on the neurite outgrowth of neuronal culture cells and

AC C

EP

TE D

M AN U

SC

RI PT

866

ACCEPTED MANUSCRIPT scopolamine-induced memory impairment in mice. Biological and Pharmaceutical Bulletin

892

24, 1434-1436.

893

Yegambaram, M., Manivannan, B., G Beach, T., U Halden, R., 2015. Role of Environmental

894

Contaminants in the Etiology of Alzheimer’s Disease: A Review. Current Alzheimer

895

Research 12, 116-146.

896

Youdim, M.B., Fridkin, M., Zheng, H., 2005. Bifunctional drug derivatives of MAO-B

897

inhibitor rasagiline and iron chelator VK-28 as a more effective approach to treatment of

898

brain ageing and ageing neurodegenerative diseases. Mechanisms of ageing and development

899

126, 317-326.

900

Yuhai, G., Zhen, Z., 2015. Significance of the changes occurring in the levels of interleukins,

901

SOD and MDA in rat pulmonary tissue following exposure to different altitudes and

902

exposure times. Experimental and therapeutic medicine 10, 915-920.

AC C

EP

TE D

M AN U

SC

RI PT

891

ACCEPTED MANUSCRIPT Figure Legends

EP

TE D

M AN U

SC

RI PT

Figure 1. Effect of scopolamine and lactucopicrin on viability of C6 glioma cells. (A) C6 cells were treated with vehicle or various concentrations (1, 3, 5, and 10 mM) of scopolamine for 1 h. After 24 h incubation in medium containing 2 % FBS, cell viability was determined by an MTT assay; it is expressed as percentage cell viability. All data in the graph are the mean ± standard deviation of at least three experiments. #P<0.05, ##P<0.01, and ###P<0.001 compared with vehicle-treated control cells. (B) C6 cells were pre-treated with 3 mM scopolamine for 1 h and then treated with vehicle or various concentrations (0.5, 1, and 2 µM) of lactucopicrin for 24 h. Galantamine (2 µM) was used as a positive control. ##P<0.01 compared with vehicle-treated control cells, *P<0.05, **P<0.01 compared with cells only pre-treated with scopolamine. (C) C6 cells were treated with scopolamine, lactucopicrin, and galantamine as described previously. After 24 h treatment, the cells were analyzed for apoptosis using Annexin V-FITC dye and flow cytometry. The number of apoptotic events was based on the number of early and late apoptotic cells (lower right and upper right quadrant, respectively). (D) Percent apoptosis was determined by calculating the concentration of early (Annexin V) and late (Annexin V+PI) apoptotic cells. ###P<0.001 compared with vehicle-treated control cells; **P<0.01 and ***P<0.001 compared with scopolamine pre-treated cells. (E) Cells were treated as described in (B). Cell lysates were separated using SDS-PAGE and the protein levels of intact caspase 3, cleaved caspase 3, and α-tubulin as an internal control were analyzed by Western blot. (F) The optical densitometry data of each band in (E) were normalized to α-tubulin and the ratio of cleaved caspase 3 to total caspase 3 was plotted as the mean ± standard deviation of at least three experiments. ##P<0.01 compared with vehicle-treated control cells; **P<0.01 and ***P<0.001 compared with cells only pre-treated with scopolamine.

AC C

903 904 905 906 907 908 909 910 911 912 913 914 915 916 917 918 919 920 921 922 923 924 925 926 927

ACCEPTED MANUSCRIPT

EP

TE D

M AN U

SC

RI PT

Figure 2. Effect of scopolamine and lactucopicrin on ROS accumulation in C6 glioma cells. (A) C6 cells were pre-treated with vehicle (a) or 3 mM scopolamine for 1 h (b) and then treated with 0.5, 1, and 2 µM lactucopicrin (c, d, and e, respectively) or 2 µM galantamine (f) for 24 h. ROS levels were visualized using DCFH-DA staining and fluorescence microscopy. DCF fluorescence intensity is an indication of the amount of ROS present in the cells. (B) Percent ROS was calculated from DCF fluorescence intensity and is plotted as the mean ± standard deviation of at least three experiments. ##P<0.01 compared with vehicle-treated control cells; **P<0.01 and ***P<0.001 compared with cells only pre-treated with scopolamine.

AC C

928 929 930 931 932 933 934 935 936

ACCEPTED MANUSCRIPT

EP

TE D

M AN U

SC

RI PT

Figure 3. Effect of lactucopicrin on scopolamine-induced mitochondrial membrane potential (MMP) loss and cytochrome C release in C6 glioma cells. (A) C6 cells were pretreated with vehicle or 3 mM scopolamine for 1 h and then treated with various concentrations (0.5, 1, and 2 µM) of lactucopicrin or 2 µM galantamine as a positive control for 24 h. Cells were stained with rhodamine 123, and fluorescence was detected by confocal microscopy. (B) C6 cells were treated as described in (A). Lysates from the mitochondrial and cytosolic fractions were separated by SDS-PAGE and protein levels of cytosolic and mitochondrial cytochrome C (cyto C) and α-tubulin as an internal control were measured by Western blot analysis. (C) Mitochondrial and cytosolic cytochrome C bands were normalized to the level of α-tubulin in each lane and the fold increase in mitochondrial vs. cytosolic cytochrome C is shown in a bar graph. All data in the graph represents the mean ± standard deviation of at least three experiments. #P<0.05 compared with vehicle-treated control cells; * P<0.05 compared with cells only pre-treated with scopolamine.

AC C

937 938 939 940 941 942 943 944 945 946 947 948 949

ACCEPTED MANUSCRIPT

EP

TE D

M AN U

SC

RI PT

Figure 4. Effect of lactucopicrin on mAChR, p-Akt, BcL-2, and Bax in C6 glioma cells. C6 cells were pre-treated with vehicle or 3 mM scopolamine and then treated with lactucopicrin or galantamine for 24 h. (A) Cell lysates were separated by SDS-PAGE and protein levels were analyzed by Western blot using antibodies against mAChR, AKT, p-Akt, BcL-2, Bax, and α-tubulin as an internal control. (B) Band intensity was normalized to the level of α-tubulin in each lane and fold increase is shown for mAChR, p-AKT/AKT, and BcL-2/Bax. All data represent the mean ± standard deviation of at least three experiments. #P<0.05 compared with vehicle-treated control cells; *P<0.05, **P<0.01, and ***P<0.001 compared with cells only pre-treated with scopolamine.

AC C

950 951 952 953 954 955 956 957 958

ACCEPTED MANUSCRIPT

EP

TE D

M AN U

SC

RI PT

Figure 5. Effect of lactucopicrin on expression of antioxidant enzymes and NRF2. In scopolamine-induced neurotoxicity, the level of antioxidant enzymes (SOD, GPx, and catalase) decreased; these were increased significantly by lactucopicrin treatment. SOD (A), GPx (B), and catalase (C) activity in 50 µg protein from C6 cell lysates. The levels of antioxidant enzymes were compared with those in the galantamine and scopolamine control groups. (D) Cells were treated with different concentrations of scopolamine and the levels of the transcription factor NRF2 were assessed using Western blot. (E) NRF2 bands were normalized to α-tubulin and decrease in the expression of NRF2 is shown in the bar graph. NRF2 levels in C6 cells treated with lactucopicrin or galantamine after 1 h pre-treatment with 3 mM scopolamine (F) and 5 mM scopolamine (H), assayed using Western blot. NRF2 band intensity was normalized to that of α-tubulin and fold increase in NRF2 level is shown in the bar graph for 3 mM scopolamine (G) and 5 mM scopolamine (I). All data represent the mean ± SD of three experiments. #P<0.05, ##P<0.01, and ###P<0.001 compared with vehicle-treated control cells; *P<0.05 and **P<0.01 compared with cells only pre-treated with scopolamine.

AC C

959 960 961 962 963 964 965 966 967 968 969 970 971 972

ACCEPTED MANUSCRIPT

EP

TE D

M AN U

SC

RI PT

Figure 6. Effect of lactucopicrin and scopolamine on NGF secretion in C6 glioma cells and neurite outgrowth in N2a neuroblastoma cells. (A) C6 glioma cells were seeded in 24well plates and treated with lactucopicrin or galantamine as described previously. After 24 h treatment, the supernatant was collected and analyzed for NGF secretion using a β-NGF ELISA kit. NGF levels are expressed as pg/ml and data represent the mean ± standard deviation of at least three experiments. (B) N2a cells were seeded in 24-well plates and treated with lactucopicrin and galantamine for 24 h as in (A). Retinoic acid and galantamine were used as positive controls for neurite outgrowth. Automated images were captured using Essen IncuCyte and analyzed for neurite outgrowth. (C) The percent neurite outgrowth was calculated and plotted as the mean ± standard deviation of at least three experiments. #P<0.05 compared with vehicle-treated control cells; *P<0.05 and **P<0.01 compared with cells only pre-treated with scopolamine.

AC C

973 974 975 976 977 978 979 980 981 982 983 984

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT

4 5 6

Highlights
Lactucopicrin prevents scopolamine-induced apoptosis by blocking caspase 3 cleavage.
Scopolamine-induced C6 cell toxicity by ROS generation, considered as valid neurodegeneration model.

RI PT

1 2 3

7


Lactucopicrin recovers mAChR and antiapoptotic proteins expression.

8


Lactucopicrin enhance ∆ψm, NRF2- mediated antioxidative enzyme and inhibits cyto

SC

TE D

M AN U


Induces NGF secretion and neurite outgrowth by lactucopicrin treatment in Scopolamine model.

EP

10 11

C release in scopolamine model.

AC C

9