Stereoisomers of Schisandrin B Are Potent ATP ... - ACS Publications

Subscriber access provided by NEW MEXICO STATE UNIV

Article

Stereoisomers of Schisandrin B Are Potent ATP Competitive GSK-3# Inhibitors with Neuroprotective Effects against Alzheimer’s disease: Stereochemistry and Biological Activity Xiaolong Hu, Cui Guo, Ji-Qin Hou, Jia-Hao Feng, Xiao-Qi Zhang, Fei Xiong, Wen-Cai Ye, and Hao Wang ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.8b00252 • Publication Date (Web): 26 Jun 2018 Downloaded from http://pubs.acs.org on July 1, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 39 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Chemical Neuroscience

1

Stereoisomers of Schisandrin B Are Potent ATP Competitive GSK-3β Inhibitors with

2

Neuroprotective Effects against Alzheimer’s disease: Stereochemistry and Biological Activity

3 4

Xiao-Long Hu†, Cui Guo†, Ji-Qin Hou†, Jia-Hao Feng†, Xiao-Qi Zhang||, Fei Xiong‡, Wen-Cai Ye||,

5

and Hao Wang †, *

6

†

7

Traditional Chinese Pharmacy. China Pharmaceutical University, Nanjing 210009, People’s Republic

8

of China.

9

‡

State Key Laboratory of Natural Medicines, Department of TCMs Pharmaceuticals, School of

State Key Laboratory of Bioelectronics, Jiangsu Laboratory for Biomaterials and Devices,

10

Southeast University, Nanjing 210009, People’s Republic of China.

11

||

12

People’s Republic of China.

13

*

14

E-mail: [email protected]

15

Postal address: Department of TCMs Pharmaceuticals, China Pharmaceutical University, Nanjing

16

210009, People’s Republic of China.

Institute of Traditional Chinese Medicine & Natural Products, Jinan University, Guangzhou 510632,

Corresponding author: Hao Wang, Professor

17 18

1

ACS Paragon Plus Environment

ACS Chemical Neuroscience 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1

ABSTRACT

2

Glycogen synthase kinase-3β (GSK-3β) is a key enzyme in hyper-phosphorylation of tau proteins

3

and is a promising therapeutic target in Alzheimer’s disease (AD). Here, we reported, for the first

4

time, that the stereoisomers of Schisandrin B (Sch B), (+)–1, (–)–1, (+)–2, and (–)–2, were potent

5

GSK-3β inhibitors. They were demonstrated to selectively target GSK-3β in an orthosteric binding

6

mode, with IC50 values of 340, 290, 80, and 70 nM, respectively. Further study showed that these

7

stereoisomers can significantly increase the expression of p-GSK-3β (Ser9), and decrease the

8

expressions of p-GSK-3β (Tyr216) and p-GSK-3β (Tyr279). Finally, these compounds can alleviate

9

the cell injury induced by Aβ, and the cognitive disorders in AD mice, especially (+)-2 and (-)-2.

10

Collectively, the stereoisomers of Sch B, especially (+)–2 and (–)–2, were found to be potential

11

selective ATP-competitive GSK-3β inhibitors, which further affected their anti-AD effects. These

12

promising findings explained the biological target of Sch B in AD, and bring a new understanding in

13

the stereochemistry and bioactivities of Sch B.

14 15 16

Keywords: Schisandrin B; stereoisomers; GSK-3β inhibitors; Alzheimer’s disease; SH-SY5Y cells

17 18 19

2


Page 2 of 39

Page 3 of 39 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1


1. INTRODUCTION

2

Alzheimer’s disease (AD), the most common form of dementia, is a progressive brain disorder

3

that affects memory, thinking, and language skills. Hyper-phosphorylation of tau proteins, which

4

results in neurofibrillary tangles, plays a critical role in AD pathology. Evidence indicate that

5

glycogen synthase kinase-3β (GSK-3β) is a key enzyme in hyperphosphorylation of tau proteins and

6

is a promising therapeutic target in AD.1,2 Recently, many GSK-3β inhibitors and synthetic

7

compounds, such as maleimides, amino pyrazoles, thiazoles, and 2,4-disubstituted thiadiazolidinones,

8

were designed, and several are currently in clinical trials. 3 However, most of these inhibitors exhibit

9

cell toxicity, poor selectivity toward GSK-3β, and low blood-brain barrier (BBB) penetration, which

10

severely affect their clinical application. 4 Traditional Chinese medicine (TCM) has demonstrated its

11

effectiveness in treating nerve damage diseases over its long history of empirical use. Over the past

12

decade, various databases of natural compounds derived from TCM plants have been developed to

13

provide information about new lead compounds against AD. 5 For example, ginsenoside Rb1, which

14

is isolated from Panax ginseng and a famous TCM herb with anti-aging efficacy, shows potent

15

inhibitory effects on GSK-3β.

16

commonly used as a brain-strengthening drug, revealed inhibition of tau phosphorylation by

17

suppressing GSK-3β. 7 Therefore, TCMs and their bioactive ingredients provide a valuable database

18

for drug screening and development of treatment for AD.

6

Onjisaponin, which is derived from Polygala tenuifolia and is

19

The fruits of Schisandra chinensis (Turcz.) Baill. (Wuweizi), a famous TCM herb, are

20

commonly used in treating mental illness and memory loss. 8 Single herb preparations of S. chinensis,

21

such as “Wuweizisyrup” and “Wuweizi tablet”, are clinically approved by the State Food and Drug

22

Administration of China for treating depression and amnesia.

23

conducted to confirm the active ingredients and specific agents in this TCM plant. Many reports have

24

recently demonstrated that dibenzo-cyclooctadiene lignans are the main active components of S.

25

chinensis,

26

dibenzo-cyclooctadiene lignans, schisandrin B (Sch B) is one of the most abundant,

27

compound has been found to possess anti-AD effects in Aβ-induced dementia model of rats. 13 Sch B

28

has also been demonstrated to exert several biological effects, including anti-oxidant,

29

anti-inflammation, 15 and neuroprotective effect,

10,11

9

Thus, in-depth studies should be

which is applied in cognitive disorders using models of dementia. Among

16

12

and this

14

which also might be contributed to its anti-AD

3



Page 4 of 39

1

activities. In addition, a previous study reported that Sch B features low toxicity and can cross BBB

2

with a complete pharmacokinetics process in the brain. 17,18 These related findings make Sch B a

3

potential lead compound for AD treatment. However, Sch B is a complex compound consisting of

4

several stereoisomers.

5

effects of its stereoisomers were not clarified till now. In fact, stereochemical factors are known to

6

play a significant role in the metabolism of drugs and biological activities.

7

necessary to investigate the biological differences in the stereoisomers of Sch B for the following

8

drug development.

9

19

The biological target of Sch B and the differences among the anti-AD

20,21

Therefore, it is

In the present study, four stereoisomers of Sch B, namely, (+) γ-Schisandrin [(+)–1], 12

22

23

12

(–)

10

γ-Schisandrin [(–)–1],

11

isolated from the fruits of S. chinensis by preparative high-performance liquid chromatography

12

(HPLC) and chiral HPLC separations. Their absolute configurations were first confirmed by

13

single-crystal X-ray diffraction analyses using Cu Kα radiation. The potential of Sch B stereoisomers

14

as GSK-3β inhibitors was revealed for the first time. Notably, we observed the significant differences

15

in the inhibitory effects of these stereoisomers against GSK-3β. These differences were reflected in

16

the following in vitro and in vivo studies, indicating that GSK-3β inhibition of these stereoisomers is

17

the main mechanism governing their anti-AD effects.

18

2. RESULTS AND DISCUSSION

Kadsuranin [(+)–2],

and Gomisin N [(–)–2],

were simultaneously

19

Previous studies have demonstrated that Sch B exerts anti-AD effects and performs many

20

biological activities, including anti-oxidation, anti-inflammation, and neuroprotective actions.

21

However, these studies commonly used the Sch B complex, and no report clarified its biological

22

target, affecting its further development and utilization. In the present study, four stereoisomers of

23

Sch B, (+)–1, (–)–1, (+)–2, and (–)–2, were simultaneously isolated from the fruits of S. chinensis by

24

means of the combination of preparative HPLC and chiral HPLC techniques, and their structures and

25

absolute configurations were firstly confirmed by means of single-crystal X-ray crystallography

26

analyses. Furthermore, Sch B’s biological target, GSK-3β, and the bioactivity differences of the four

27

stereoisomers were demonstrated for the first time.

28

2.1. Molecular docking study. Molecular docking allows evaluation of the interaction and binding

29

energy of protein-ligand complexes. All those compounds have displayed a higher CDOCKER 4


Page 5 of 39 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

interaction energy than the reference compound, 6-bromoindirubin-3’-oxime (BIO). and the ligands

2

that satisfied the necessary interactions with the active site compounds were chosen, Table 1.

3

Interaction residues analysis: (+)-1 can bind to Val 135 and Lys 85 by H-bond, and bind to Leu 188,

4

Cys 199, and Val 70 by π-bond; (-)-1 can bind to Val 135 and Lys 85 by H-bond, and bind to Val 70,

5

and Cys 199 by π-bond; (+)-2 can bind to Arg 141, Ile 62, Glu 137, Val 135, and Pro 136 by H-bond,

6

and bind to Ile 62, Leu 188, and Val 70 and Leu 188 by π-bond; (-)-2 can bind to Arg 141, Ile 62, Glu

7

137, Val 135, and Pro 136 by H-bond, and bind to Ile 62, Cys 199, Val 70, and Leu 188 by π-bond

8

(Figure 3). Previous study has demonstrated that Val135, Glu137, Arg141, and Cys199 were the key

9

residues to regulate the activity of GSK-3β.

24

From the above results, it was easy to see that (+)-2

10

and (-)-2 can form more H-bonds with these key residues than (+)-1 and (-)-1. Structurally, whether

11

(+)-1 or (-)-1, C7-CH3 and C8-CH3 are respectively in equatorial orientation and axial orientation,

12

while (+)-2 or (-)-2 are the opposite. Combined with docking study, the conformation of (+)-2 or

13

(-)-2 was more conducive to forming interactions with GSK-3β. These findings make the potential of

14

Sch B as GSK-3β inhibitors, and prompt the differences of these stereoisomers in GSK-3β inhibition

15

(2 > 1).

16

2.2. Inhibitory effects of (+)–1, (–)–1, (+)–2, and (–)–2 on GSK-3β. To corroborate the docking

17

predictions, fluorescence resonance energy transfer (FRET) 25 was used to detect the effects of (+)–1,

18

(–)–1, (+)–2, and (–)–2 on GSK-3β activities, and Bio was also as the positive control. All the test

19

compounds exhibited potent inhibitory effects with IC50 values at nanomolar grade (Table 1). The

20

results of GSK-3β inhibition of above compounds followed a similar order of size with the docking

21

scores, further suggesting that (+)–1, (–)–1, (+)–2, and (–)–2 targeted GSK-3β. Kinetic experiments

22

suggested that these compounds act as both substrate and ATP-competitive GSK-3β inhibitors

23

(Figure 4).

24

2.3. Selectivity of protein kinase inhibition. Drug design strategies for GSK-3β inhibitors can be

25

grouped mainly into (i) metal-competitive inhibition, (ii) ATP-competitive inhibition, (iii) inhibition

26

by binding at catalytic triad, (iv) irreversible inhibition, (v) substrate-competitive inhibition, (vi)

27

peptide-like inhibitors, and (vii) allosteric inhibition.

28

inhibition, the ATP-competitive strategy has been extensively explored and reported. However, most

29

ATP-competitive inhibitors failed at the preclinical stage, mainly owing to selectivity issues against

4

Among the seven strategies of GSK-3β

5



Page 6 of 39

1

CDK-2 and other phylogenetically related kinases, which were further attributed to the close

2

homology of ATP binding sites of these enzymes. 26 Therefore, the selectivity of (+)–1, (–)–1, (+)–2,

3

and (–)–2 on GSK-3β bear significance in subsequent clinical drug development. CDK2 is the

4

closest kinase to GSK-3β from a homology perspective (overall 33% amino acid identity),

5

most reported GSK-3β inhibitors also potently inhibit CDK2. 28 However, once CDK2 was inhibited,

6

cell cycle and cellular proliferation were disrupted, which resulted in toxicological effects of

7

GSK-3β inhibition in GSK-3β-related pathologies.

8

(GSK-3β)

9

GSK-3β. Moreover GSK-3 exists in two isoforms, GSK-3α (51 kDa) and GSK-3β (47 kDa), which

29

Our results showed that the IC50

27

and

(CDK2)/IC50

reached over 100 (Table 2), suggesting that (+)–1, (–)–1, (+)–2, and (–)–2 selectively targeted

30

10

share 84% overall identity and 98% identity within their respective catalytic domains.

11

primary isoforms are non-specifically expressed, with high levels observed in the brain. 31 GSK-3β is

12

the predominant isoform in most brain areas.

32

13

kinase required for tau hyperphosphorylatio.

33,34

14

and GSK-3β share very similar functions in several cellular processes. Our results showed a certain

15

degree of difference in the selectivities (about 10-fold) of GSK-3α/β for these compounds (Table 2).

16

These compounds were also tested for selectivity against a panel of 10 human protein kinases, which

17

were selected from homologous kinases. Results displayed the weak inhibitory effects of (+)–1, (–)–

18

1, (+)–2, and (–)–2 over other homologous kinases (Table 2). These promising findings

19

demonstrated that (+)–1, (–)–1, (+)–2, and (–)–2 are highly selective GSK-3β inhibitors.

20

2.4. Effects of (+)–1, (–)–1, (+)–2, and (–)–2 on expression of p-GSK-3β/p-tau and level of hepatic

21

glycogen in vivo. All the studies mentioned above demonstrated the potential of these stereoisomers

22

as in vitro GSK-3β inhibitors. However, whether they can produce the same biological effects in vivo

23

remain unknown. Evidence showed that GSK-3β inhibitors can increase the expression of p-GSK-3β

24

(Ser9), the active form of GSK-3β, and in turn decrease the expression of p-tau in hippocampus.

25

Therefore, ICR mice were used to detect the expressions of p-GSK-3β and p-tau, and the level of

26

hepatic glycogen after drug treatment. Our results showed that (+)–1, (–)–1, (+)–2, and (–)–2 exert

27

similar effects on the expressions of p-GSK-3β(Ser9) and p-tau (Thr231). A more notable

28

effectiveness was observed for (+)–2 and (–)–2 (Figures 5A, 5B, 5C, and 5D). GSK-3β plays a

29

critical role in controlling hepatic glycogen. Suppression of GSK-3β activity by insulin or small

Both

Evidence suggested that GSK-3β isoform is the key However, several studies suggested that GSK-3α

6


35

Page 7 of 39 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


36,37

1

molecule inhibitors increases glycogen storage in the liver.

2

considered a testing index for verifying the inhibitory effects of these compounds on GSK-3β in vivo.

3

Figure 5E shows that (+)–1, (–)–1, (+)–2, and (–)–2, especially (+)–2 and (–)–2, significantly

4

increased the liver glycogen level. All these results suggest that (+)–1, (–)–1, (+)–2, and (–)–2,

5

especially (+)–2 and (–)–2, exhibit potential inhibitory effects on GSK-3β in vivo.

6

2.5. Effects of (+)–1, (–)–1, (+)–2, and (–)–2 on the pathways of p-GSK-3β/p-tau and neuronal

7

apoptosis. The mentioned results have confirmed (+)–1, (–)–1, (+)–2, and (–)–2 as potent GSK-3β

8

inhibitors both in vitro and in vivo and shown the significant difference between 1 and 2. The

9

anti-AD effects of these compounds in vitro and in vivo should be verified. Increasing evidence have

10

suggested that Aβ can induce GSK-3β activation in vitro and in vivo, leading to

11

hyper-phosphorylation of tau and neuronal apoptosis. 38 The Aβ-induced SH-SY5Y cell injury model

12

is commonly used as an AD model in vitro. Our study confirmed that these compounds can alleviate

13

cell injury induced by Aβ (Figures 6A and 6B) and inhibit p-GSK-3β/p-tau pathway (Figures 6C,

14

6D, 6E and 6F), whereas (+)–2 and (–)–2 retained better activities.

15

2.6. Effects of (±)–1 and (±)–2 on learning and memory abilities of APP/PS1 double transgenic

16

mice in Morris water maze test (MWM). APP/PS1 double transgenic mice were used to evaluate the

17

anti-AD effects of these compounds in vivo. The above results have demonstrated that the

18

effectiveness between (+)–1 and (–)–1, as well as between (+)–2 and (–)–2, were equivalent,

19

respectively. Therefore, in order to improve the efficiency and decrease the time cost, two racemic

20

forms, (±)–1 and (±)–2, were prepared for the following experiments. APP/PS1 mice reportedly

21

exhibit early appearance of amyloid plaques at the age of 4 months and significant elevation in Aβ

22

levels and cognitive deficits at the age of 6 months. 39 Considering the peculiar pathogenesis of AD

23

in APP/PS1 mice at the age of 6 months, 5-month-old APP/PS1 mice were selected for the 4-week

24

oral administration of drug. In the first 5 days, we observed the impaired ability of APP/PS1 mutant

25

mice in water maze learning compared with the wild-type (WT) group, whereas the APP/PS1 mice

26

pre-treated with (±)–1 and (±)–2 for 4 weeks revealed improved learning ability (Figure 7B). On day

27

6, the APP/PS1 double transgenic mice spent significantly less time (16.64 ± 2.96 sec) in the

28

memorized region compared with the WT group (34.84 ± 5.96 sec), suggesting the loss of memory

29

ability at this stage. However, the (±)–1 and (±)–2 groups revealed enhanced memory ability with 7


Therefore, glycogen level was also


1

spending times of 23.05 ± 3.23, and 34.2 ± 3.23 sec, respectively (Figure 7C). The times of the

2

APP/PS1 mice group (1.8 ± 0.21 times) across the platform were significantly less than those of the

3

WT group (4 ± 0.45 times). However, pretreatment with (±)–1 and (±)–2 increased the times across

4

the platform (3 ± 0.23, and 3.9 ± 0.43 times, resepectively) (Figure 7D). Figure 7E shows the

5

pathways of searching and swimming of one random mouse from each group. Collectively, 30 mg/kg

6

of (±)–1 and (±)–2, especially (±)–2, can significantly improve learning and memory abilities in AD

7

mice.

8

2.7. Effects of (±)–1 and (±)–2 on the level of Aβ1-42 and p-GSK-3β/tau in hippocampus of

9

APP/PS1 double transgenic mice. To further confirm the effects of (±)–1 and (±)–2 on AD mice,

10

enzyme-linked immunosorbent assay (ELISA) and Western blot assays were used to detect the level

11

of Aβ1-42 and expressions of p-GSK-3β and p-tau in the hippocampus of APP/PS1 double transgenic

12

mice. As shown in Figure 7F, the APP/PS1 double transgenic mice group showed a significantly

13

increasing level of Aβ1-42 (1104 ± 54.18 pg/mL) compared with the WT group (695 ± 10.01 pg/mL).

14

However, pretreatment with 30 mg/kg of (±)–1 and (±)–2 decreased the level of Aβ1-42 (930.3 ±

15

13.87 pg/mL, and 732.9 ± 25.42 pg/mL, respectively). The expression of p-GSK-3β (Ser9) was

16

maintained at a low level in the APP/PS1 double transgenic mice group compared with the WT

17

group. However, (±)–1 and (±)–2 significantly increased the expression of p-GSK-3β. Similarly, the

18

expression of p-tau remained at a high level in the APP/PS1 double transgenic mice group compared

19

with the WT group. However, (±)–1 and (±)–2 significantly decreased the expression of p-tau

20

(Figure 7G). These results further demonstrated that the anti-AD effects of Sch B were involved in

21

GSK-3β inhibition.

22

3.

CONCLUSIONS

23

In summary, for the first time, the stereoisomers of Sch B, (+)–1, (–)–1, (+)–2, and (–)–2, were

24

subjected to single-crystal X-ray diffraction analyses using Cu Kα radiation. Notably, Sch B

25

stereoisomers, especially (+)–2 and (–)–2, were found to be potent ATP-competitive GSK-3β

26

inhibitors with efficacy in modulation of the p-GSK-3β/tau pathway and glycogen synthase level in

27

vitro and in vivo. Further study of these compounds demonstrated the stronger effects of (+)–2 and

28

(–)–2 in both the cell and APP/PS1 mice models of AD. All these findings indicate that Sch B

29

stereoisomers, especially (+)–2 and (–)–2, constitute a promising family of pharmacological 8


Page 8 of 39

Page 9 of 39 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

inhibitors for the development of novel AD therapeutic agents through GSK-3β inhibition, also bring

2

a new understanding in the stereochemistry and bioactivities of Sch B.

3

9



1

4.

2

4.1. Chemistry. The dried fruits of S. chinensis (1 kg) were powdered and extracted with 95 %

3

ethanol (3× 8 L) under reflux (3 × 2 h). The combined ethanol extracts were concentrated under

4

reduced pressure to afford a residue (170 g), which was suspended in H2O and partitioned with

5

EtOAc (3 × 400 mL). The EtOAc extract (100 g) was subjected to silica gel column chromatography

6

(200-300 mesh, 1 Kg), eluted with PE-EtOAc (98:2-90:10, v/v) to afford 5 fractions. Fraction 4 (2.16

7

g) was purified by preparative HPLC (methanol-water (80: 20, v/v, 15 mL/min) to afford Compound

8

(±)-1 (209 mg), and (±)-2 (846 mg). Compounds (±)-1, and (±)-2 was further separated into two

9

pairs of enantiomers, (+)-1 (45 mg), (-)-1 (36 mg), (+)-2 (64 mg), and (-)-2 (106mg) (Figures 1 and

10

2), by Daicel Chiralcel OD-RH semi-preparative HPLC column (250 × 10 mm, 5 µm), using

11

MeOH-H2O (90:10, 4 mL/min) as the mobile phase, respectively. The purity of all compounds is

12

over 98 %.

13

4.1.1. (+) γ-Schizandrin, (+)-1: white prisms (MeOH); [α]D25 +51.7˚ (c 0.120, MeOH); UV (MeOH)

14

λmax (log ε) 221 (4.49) nm; IR (KBr) νmax 1617, 1596 cm−1; CD (MeOH) λmax (∆ε) 218 (-38.84), 253

15

(+38.42) nm; (+)-HR-ESI-MS m/z =401.1963 (calcd. for [C23H28O6 + H]+: 401.1959); 1H NMR

16

(CDCl3, 300MHz):δH 6.56(1H, s, H-4), 6.50(1H, s, H-11), 5.97(2H, s, OCH2O), 3.56(3H, s, 1-OCH3),

17

3.84(3H, s, 14-OCH3), 3.90(H, s, 3-OCH3), 3.91(3H, s, 2-OCH3), 2.58(1H, dd, J = 13.5, 7.2 Hz,

18

H-9a), 2.45(1H, dd, J = 13.5, 1.8 Hz, H-9b), 2.33(1H, dd, J = 13.2, 9.6 Hz, H-6a), 2.06(1H, brd, 13.2,

19

H-6b), 1.80(1H, m, H-7), 1.90(1H, m, H-8), 0.76(3H, d, J = 7.2Hz, 17-CH3), 1.02(3H, d, J =7.2 Hz,

20

18-CH3); 13C NMR (CDCl3, 75MHz):δC 151.5(C-1), 139.7(C-2), 152.8(C-3), 107.4(C-4), 139.4(C-5),

21

35.4(C-6), 40.8(C-7), 33.8(C-8), 38.9(C-9), 132.5(C-10), 105.9(C-11), 147.6(C-12), 134.8(C-13),

22

141.2(C-14), 122.2(C-15), 122.4(C-16), 12.4(C-17), 21.9(C-18), 61.0(3-OCH3), 60.5(1-OCH3),

23

59.6(14-OCH3), 55.9(2-OCH3), 100.7 (OCH2O). The absolute configuration of (+)-1 was confirmed

24

by a single crystal X-ray diffraction analysis using Cu Kα radiation with the Flack parameter [0.07

25

(8)].

26

4.1.2. (-) γ-Schizandrin, (-)-1: white prisms (MeOH); [α]D25 -54.6˚ (c 0.108, MeOH); UV (MeOH)

27

λmax (log ε) 222 (4.49) nm; IR (KBr) νmax 1617, 1596 cm−1; CD (MeOH) λmax (∆ε) 218 (+42.18), 253

28

(-41.68) nm; (+)-HR-ESI-MS m/z =401.1959 (calcd. for [C23H28O6 + H]+: 401.1959); 1H NMR

29

(CDCl3, 300MHz): δH 6.57(1H, s, H-4), 6.50(1H, s, H-11), 5.97(2H, s, OCH2O), 3.56(3H, s,

METHODS

10


Page 10 of 39

Page 11 of 39 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

1-OCH3), 3.84(3H, s, 14-OCH3), 3.90(H, s, 3-OCH3), 3.91(3H, s, 2-OCH3), 2.58(1H, dd, J = 13.5,

2

7.2 Hz, H-9a ), 2.45(1H, dd, J = 13.5, 1.8 Hz, H-9b ), 2.33(1H, dd, J = 13.2, 9.6 Hz, H-6a), 2.06(1H,

3

brd, 13.2, H-6b), 1.80(1H, m, H-7), 1.90(1H, m, H-8), 0.76(3H, d, J = 7.2Hz, 17-CH3), 1.02(3H, d, J

4

=7.2 Hz, 18-CH3);

5

139.4(C-5), 35.4(C-6), 40.8(C-7), 33.8(C-8), 38.9(C-9), 132.5(C-10), 105.9(C-11), 147.6(C-12),

6

134.8(C-13), 141.2(C-14), 122.2(C-15), 122.4(C-16), 12.4(C-17), 21.9(C-18), 61.0(3-OCH3),

7

60.5(1-OCH3), 59.6(14-OCH3), 55.9(2-OCH3), 100.7(OCH2O). The absolute configuration of (-)-1

8

was confirmed by a single crystal X-ray diffraction analysis using Cu Kα radiation with the Flack

9

parameter [-0.09 (4)].

13

C NMR (CDCl3, 75MHz): δC 151.5(C-1), 139.7(C-2), 152.8(C-3), 107.4(C-4),

10

4.1.3. kadsuranin, (+)-2: white prisms (MeOH); [α]D25 +53.9˚ (c 0.128, MeOH); UV (MeOH) λmax

11

(lg ε) 227 (4.51) nm; IR (KBr) νmax 1618, 1595 cm−1; CD (MeOH) λmax (∆ε) 218 (-47.04), 251

12

(+42.83) nm; (+)-HR-ESI-MS m/z =401.1960 (calcd. for [C23H28O6 + H]+: 401.1959); 1H NMR

13

(CDCl3, 300MHz): δH 6.57(1H, s, H-4), 6.49(1H, s, H-11), 5.96(2H, s, OCH2O), 3.56(3H, s, 3-OCH3),

14

3.83(3H, s, 14-OCH3), 3.90(H, s, 1-OCH3), 3.91(3H, s, 2-OCH3), 2.56(2H, m, H-6), 2.25(1H, dd, J =

15

13.2, 9.3 Hz, H-9b), 2.04(1H, brd, J = 12.9 Hz, H-9a), 1.90(1H, m, H-7), 1.80(1H, m, H-8), 0.75(3H,

16

d, J = 6.9 Hz, 18-CH3), 0.99(3H, d, J =7.2 Hz, 17-CH3); 13C NMR (CDCl3, 75MHz): δC 151.6(C-1),

17

140.1(C-2), 151.5(C-3), 110.7(C-4), 134.1(C-5), 39.1(C-6), 33.5(C-7), 40.7(C-8), 35.5(C-9),

18

137.8(C-10), 102.9(C-11), 148.6(C-12), 134.5(C-13), 141.1(C-14), 121.4(C-15), 123.3(C-16),

19

21.5(C-17),

20

100.7(OCH2O). The absolute configuration of (+)-2 was confirmed by a single crystal X-ray

21

diffraction analysis using Cu Kα radiation with the Flack parameter [-0.01 (4)].

22

4.1.4. gomisin N, (-)-2: white prisms (MeOH); [α]D25 -52.6˚ (c 0.116, MeOH); UV (MeOH) λmax (lg ε)

23

223 (4.50) nm; IR (KBr) νmax 1618, 1596, 1570 cm−1; CD (MeOH) λmax (∆ε) 218 (+47.11),

24

251(-42.68) nm; (+)-HR-ESI-MS m/z =401.1965 (calcd. for [C23H28O6 + H]+: 401.1959); 1H NMR

25

(CDCl3, 300MHz): δH 6.57(1H, s, H-4), 6.49(H, s, H-11), 5.96(2H, s, OCH2O), 3.56(3H, s, 3-OCH3),

26

3.83(3H, s, 14-OCH3), 3.90(H, s, 1-OCH3), 3.91(3H, s, 2-OCH3), 2.57( 2H, m, H-6 ), 2.25(1H, dd, J

27

= 13.2, 9.3Hz, H-9b), 2.04 (1H, brd, J = 12.9 Hz, H-9a), 1.90(1H, m, H-7), 1.80(1H, m, H-8),

28

0.75(3H, d, J = 7.2 Hz, 18-CH3), 0.99( 3H, d, J =7.2 Hz, 17-CH3); 13C NMR (CDCl3, 75MHz): δC

29

151.6(C-1), 140.1(C-2), 151.6(C-3), 110.7(C-4), 134.1(C-5), 39.1(C-6), 33.5(C-7), 40.7(C-8),

12.8(C-18),

61.0(2-OCH3),

60.5(3-OCH3),

11


59.6(14-OCH3),

55.9(1-OCH3),


Page 12 of 39

1

35.5(C-9), 137.8(C-10), 102.9(C-11), 148.6(C-12), 134.5(C-13), 141.1(C-14), 121.4(C-15),

2

123.3(C-16), 21.5(C-17), 12.8(C-18 ), 61.0(2-OCH3), 60.5(3-OCH3), 59.6(14-OCH3), 55.9(1-OCH3),

3

100.7 (OCH2O). The absolute configuration of (-)-2 was confirmed by a single crystal X-ray

4

diffraction

5

All the 1H- and 13C-NMR signals of the compounds were assigned with the aid of

6

COSY, HSQC and HMBC experiments.

7

4.2. Biological assays

8

4.2.1. Generals. GSK-3β kinase activity kit and Hoechst 33342 staining kit were purchased from

9

Invitrogen (Carlsbad, CA, USA). The prephosphorylated polypeptide substrate GS-2 was

10

synthesized by GL Biochem Ltd. (Shanghai, China). ATP·2Na was purchased from Sigma-Aldrich

11

(St. Louis, MO). Annexin V-PI double staining kit was purchased from BD Pharmingen (Carlsbad,

12

CA, USA). MTT, 0.25 % trypsin-EDTA and dimethyl sulfoxide (DMSO) were purchased from

13

Amresco (Solon, OH, USA). PVDF transfer membrane was purchased from Millipore Corp (Bedford,

14

MA, USA). Lactate Dehydrogenase (LDH) kit and MDC staining kit were purchased from Nanjing

15

Key-Gen Biotech (Nanjing, China). Liver glycogen kit was purchased from Cusabio biotech Co., Ltd

16

(Wuhan, China). Aβ25-35 and 6-bromoindirubin-3-oxime (Bio) were purchased from Sigma-Aldrich

17

(St. Louis, MO, USA). Rabbit antibodies for GSK-3β, p-GSK-3β (Ser9), p-GSK-3β (Tyr216),

18

p-GSK-3β (Tyr279),

19

IgG labeled with Alexa Fluor-568 were purchased from Cell Signalling Technology (Beverly, MA,

20

USA). Mouse antibodies for Tau, p-Tau (Ser396), p-Tau (Thr231), β-actin, and HRP-conjugated goat

21

anti-rabbit (mouse) IgG were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). All

22

remaining chemicals and reagents used in this experiment were the highest available analytic grade.

23

4.2.2. Docking assay. The chemical structures of test compounds were sketched in Chemdraw Ultra

24

(7.0), and saved in Mol2 format. X-ray crystal structure of GSK-3β (PDB ID: 1UV5, resolution = 2.8

25

Å)

26

software was used to prepare the protein required in docking. Ligands and water molecules were

27

removed from the crystal structures of the protein, and hydrogen atoms were added. Charges were

28

assigned to the atoms according to the method of AMBER7FF99. A short minimization (100 steepest

29

descent steps with tripos force field) was performed to release internal strain. This docking engine

40

analysis

using

Cu

Kα

radiation

with

the

Flack

parameter

[0.03

(4)]. 1

H-1H

Bax, and Bcl-2, anti-mouse IgG labeled with Alexa Fluor-488, and anti-rabbit

was obtained from the Protein Data Bank (PDB) (http://www.wwpdb.org). The sybyl 6.9.1

12


Page 13 of 39 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

takes a multi-conformer database of one or more ligands, a target protein structure, a box defining

2

the active site of the protein based on the co-crystallized ligand, and several optional parameters as

3

input. The ligand conformers and protein structure are treated as rigid during the docking process.

4

CDOCKER, a docking module, was used to simulate the binding style between GSK-3β and

5

compounds. All the parameters were set as the default mode. A 3D diagram of their interaction is

6

manifested to confirm, and their docking pose was presented for the analysis of the interactions,

7

including hydrogen bond, hydrophobic bond, π-π interaction and so on. The prepared protein and the

8

ligands were imported onto DS v4.5 for the execution of the docking protocol. Corresponding

9

results were evaluated based upon the -CDOCKER interaction energy, hydrogen bond interaction,

10

and the binding mode pattern.

11

4.2.3. In-vitro GSK-3β kinase assay with cell-free model. The enzymatic activity of the kinases is

12

determined with a commercial system based on the Z’-LYTE® technology, using human GSK-3β

13

recombinant kinase as the enzyme source.

14

energy transfer (FRET) process between fluorescein and coumarin. The GSK-3β and ATP

15

concentration was optimized to obtain the desired percent phosphorylation with an acceptable

16

Z’-factor value, which indicates the quality of an assay; Z’-factor of 0.5 or greater classify an assay

17

as excellent. Z’-factor value of 0.92 was obtained at final kinase and ATP concentrations of 50 ng/mL

18

and 15 µM, respectively. Compounds are serially diluted in 4% DMSO (10 serial dilutions) and these

19

dilutions are further diluted 1/25 with reaction buffer. 2.5 µL of these solutions are added to the

20

reaction mixture described below so that final compound concentration in the assay ranges from 10

21

µM to 1 nM in 4 % (v/v) DMSO. The IC50 value for each experiment was obtained using nonlinear

22

regression of the log (concentration) versus percent inhibition values (Graphpad Prism 7.0). Assays

23

were conducted in triplicate, average values were calculated, and no outliers were removed.

24

4.2.4. Selectivity studies of kinases inhibition. The activities of (+)-1, (-)-1, (+)-2, and (-)-2 on the

25

other homologous kinases were screened against a panel of 11 human serine/threonine kinases by

26

Eurofins using the Eurofins Kinase Profiler Selectivity Testing Service.

27

4.2.5. Cell culture. Human neuroblastoma cell line, SH-SY5Y cells were obtained from Professor

28

Ming Yan (China Pharmaceutical University) as a present, and routinely cultured in DMEM

29

supplemented with 10 % (v/v) fetal bovine serum (Gibico) and 1 % (v/v) penicillin-streptomycin in a

25

This technology utilizes the fluorescence resonance

13



1

humidified atmosphere of 5 % CO2 at 37 °C.

2

4.2.6. Cell viability assay. SH-SY5Y cells were seeded in 96-well plate with a destiny of 5 × 103

3

cells/well. After 24 hours, the cells were pretreatment with different concentrations of (+)-1, (-)-1,

4

(+)-2, and (-)-2 (5, 10, 25 µM) or Bio (25 µM) for 4 hours and then of Aβ25-35 (final concerntration:

5

25 µM) was added to the medium (DMEM) for 24 hours. After that, MTT solution (20 µL, 0.5

6

mg/mL in DMEM) was added to each well and incubated for 4 hours at 37 °C. The medium was

7

removed and 150 µL of DMSO was added into each well. The absorbance was measured at 490 nm

8

using a microplate reader (TECAN, PRO NanoQuant).

9

4.2.7. Double-Label immunostaining assay of SH-SY5Y cells. SH-SY5Y cells were seeded in

10

6-well plate with a destiny of 2 × 105 cells/well. After 24 hours, the cells were pretreatment with

11

different concentrations of (+)-1, (-)-1, (+)-2, and (-)-2 (5, 10, 25 µM) or Bio (25 µM) for 4 hours.

12

Then, Aβ25-35 (final concerntration: 25 µM) was added to the medium (DMEM) for another 1 hour.

13

After the above treatments, cells were fixed with ice-cold 4 % paraform-aldehyde in PBS (pH 7.4)

14

for 20 minutes. Fixed cells were washed with cold PBS and were added with blocking buffer (10%

15

HS and 0.3% Triton X-100 in PBS) for 1 hour at room temperature. Cells were incubated overnight

16

at 4 ℃ in primary rabbit antibody to p-GSK-3β (Ser9) (1:400), and mouse antibody to p-Tau(Thr231)

17

(1:400) and rinsed with washing buffer (1.5 % HS and 0.1 % Triton X-100 in PBS) three times. Then,

18

cells were incubated with appropriate secondary antibodies conjugated to either Alexa488 or

19

Alexa594, diluted in blocking buffer, for 2 hours at room temperature. Cells were washed three times

20

with washing buffer, visualized with a fluorescence microscope (Nikon, Japan).

21

4.2.8. Animals. For ICR mice: Six-week-old male ICR mice was purchased from Model Animal

22

Research Center of Nanjing University with body weight averaging 25 g, fed ad libitum with

23

standard food and water, were randomly assigned into six groups (n=6 mice/cage). Group A: Normal

24

controls; Group B: Positive controls (LiCl, 10 mg/kg); Group C: (+)-1, 30 mg/kg; Group D: (-)-1, 30

25

mg/kg; Group E: (+)-2, 30 mg/kg; Group F: (-)-2, 30 mg/kg. On the day of the experiment, food and

26

water were removed 4 hours before drug administration. Then, animals were intragastrically (i.g.)

27

administrated with 30 mg/kg of (+)-1, (-)-1, (+)-2, and (-)-2, while the control group was injected

28

with the saline vehicle (i.g.). After 3 hours, mice were sacrificed, and then liver or brain was rapidly

29

collected to perform the next experiments. All protocols have been reviewed and approved by the 14


Page 14 of 39

Page 15 of 39 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

Experimental Animal Center of China Pharmaceutical University. For APP/PS1 double transgenic

2

mice (used for morris water maze test): C57BL/6J mice were obtained from Model Animal Research

3

Center of Nanjing University. Offspring were genotyped using PCR on DNA isolated from tail

4

biopsy. For behavioral testing, we used male APP/PS1 and wild type mice kept in standard animal

5

cages under conventional laboratory conditions (12 h light/dark cycle, 22 °C), and ad libitum access

6

to food and water. Experiments were conducted during the light phase of the activity cycle. The

7

APP/PS1 mice model has been shown to develop AD neuropathology at 2 months of age, whereas

8

behavioral impairments occur from 6 months of age onwards.40 Therefore, we initiated (±)-1 and

9

(±)-2 treatments at 5 months of age, when amyloid deposits have been shown to be still rather scarce

10

in APP/PS1 mice, for a period of 1 month. APP/PS1 and wild type mice were randomly assigned to

11

four groups, including group A: wild type mice, group B: APP/PS1 model mice, group C: APP/PS1

12

model mice + (±)-1 (30 mg/kg), and group D: APP/PS1 model mice + (±)-2 (30 mg/kg). All

13

protocols have been reviewed and approved by the Experimental Animal Center of China

14

Pharmaceutical University.

15

4.2.9. Determination of liver glycogen (ICR mice). Liver glycogen level of ICR mice was

16

characterized following a commercial kit by ELISA assay. Briefly, after 30 min of drug

17

administration, the livers of each group (n=6 mice/group) were removed, and homogenized (IKA-T8

18

Ultra-Turrax, Germany) in cold PBS (100 mg tissue/mL PBS) for 5 min. The homogenate was

19

centrifuged at 3000 rpm for 5 min. Then, the supernatant fluid was collected to determine the

20

concentration of proteins of each sample. After these treatments, all the samples were conducted

21

according to the specification. Finally, the absorbance was detected at 450 nm using a microplate

22

reader (TECAN, PRO NanoQuant).

23

4.2.10. Double-labeling immunostaining of hippocampus (ICR mice). After 30 min of drug

24

administration, ICR mice of each group (n=6) were anesthetized with ether for 60 sec, and then

25

perfused intracardially with saline (PH = 7.4) and 4 % paraformaldehyde (PFA) in 0.1 M sodium

26

dihydrogen phosphate containing 0.48 % sodium hydroxide (PH = 7.4). After fixative perfusion,

27

brains were removed, placed in 4 % PFA at 4 ℃ overnight, and then transferred to 30 % sucrose

28

solution till settle down. The cryoprotected brains were sectioned serially at 30 µM in the coronal

29

plane using a freezing microtome (Leica CM3050S, Germany), collected in Dulbecco’s PBS solution 15



1

containing 0.1 % sodium azide solution, and stored at 4 ℃. Brain sections were treated with 0.6 %

2

H2O2 in phosphate-buffered saline (PBS) for 10 min and then washed with Tris-buffered saline (TBS)

3

for three times. Then, sections were blocked in 0.5 % Triton-x-100 in TBS and Triton X-100/3%

4

horse serum (TBS-TS) for 1 hour at room temperature, and incubated with primary antibodies of

5

p-GSK-3β (Ser9) (Rabbit polyclonal; 1:500); the p-tau (Thr231) (Mouse polyclonal; 1:500) at 4 °C

6

for 72 hours. Afterwards, the sections were washed with TBS, and then were incubated with

7

secondary goat-rabbit IgG antibodies (1:1000) at room temperature for 2 hours. Then, the sections

8

were washed with TBS. Finally, DAB solution was added to incubate for 5 min. Images were

9

obtained with a DP72 digital camera (Nikon, Japan).

10

4.2.11. Kinetic analysis on GSK-3β. The protocol of the kinetic experiments was much similar to

11

one of GSK-3β inhibition tests. The activities of compounds (+)-1, (-)-1, (+)-2 and (-)-2 were

12

measured separately at concentrations of 1 µM and 2 µM. In the experiments for testing the

13

relationship between the two compounds and ATP, the concentration of substrate GS-2 was kept

14

unchanged at 15 µM, while the concentration of ATP was set at 6.5, 10, 15, 25, 50, 100 µM,

15

separately. Then, in the following experiments for testing the relationship between them and GS-2,

16

the concentration of ATP was kept unchanged at 15 µM, while GS-2 concentration was set at 6.5, 10,

17

15, 25, 50, 100 µM, separately.

18

4.2.12. Morris water maze test. The Morris water maze (MWM) test was used to examine the

19

changes in the learning and memory abilities of the APP/PS1 AD mice, as previously described. 41 In

20

brief, a circular water maze with the diameter was 120 cm and the height was 50 cm was used. A

21

hidden platform with a diameter of 9 cm was inside the maze and the surface was 1.0 cm below the

22

surface of the water. Floating plastic particles were placed on the surface of the water to hide the

23

platform from sight. The temperature of the water was 22.0 ± 0.5 C ̊ . The experiment was performed

24

in a double-blind manner. The individual who performed the experiment was familiar with the mice

25

from the two weeks of oral feeding. For the experiment, the mice were placed in a random area

26

inside the maze for free swimming until they identified the hidden platform. The whole experiment

27

lasted for 6 days. For the first 5 days, the mice were left in the maze to find the platform with a

28

maximum time of 90 sec. The learning section was repeated 4 times each day, with an interval of 1 h

16


Page 16 of 39

Page 17 of 39 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

between each session. On the last day, the platform was removed and the time that the mice spent in

2

the memorized region was recorded over a period of 120 sec.

3

4.2.13. Elisa assay for Aβ1-42 in hippocampus. The sample preparation was similar as previous study

4

42

5

from the skull and was dissected. First, the bilateral cerebral cortex was removed, followed by the

6

hippocampus, after which the remaining brain was placed on a glass slide over dry ice. The cerebral

7

cortex and hippocampus of each mice were rapidly dissected and flash frozen at -80 °C for next

8

studies. The frozen hippocampus tissue, which mixed with 9 times the cold saline, was homogenized

9

in a glass grinder on ice. The homogenate was placed in EP tube pretreated at 4 °C and centrifuged

10

with speed of 12, 000 r/min for 10 min. The supernatant was kept in -20 °C to prepare for testing.

11

Before the ELISA assay, BCA assay was used to determine the protein concentration. Then, the

12

insoluble Aβ1-42 level was measured in hippocampus homogenates by the sandwich ELISA method

13

and assay protocol was performed in accordance with the manufacturer’s suggestions (CUSABIO

14

BIOTECH, China). 96-well were exposed to samples for 2 hours and washed before incubation with

15

biotinylated detection antibody for 2 hours. Staining of coated wells was developed using 100 µL

16

streptavidin-HRP (1:200) for 30 min at RT and visualized with 100 µL SureBlue Reserve TMB

17

Microwell Peroxidase Substrate. 100 µL TMB Stop solution was added to stop the reaction and

18

plates were read on a SpectraMax plate reader set at 450 nm with wavelength correction at 540 nm.

19

4.2.14. Statistical analysis. The in vitro data are shown as mean ± SD and in vivo results as mean ±

20

SEM. Statistical analysis was performed in all the experiments shown using the Graphpad Prism 7.0

21

software (GraphPad Software Inc, La Jolla, CA, USA). Statistical analysis for multiple groups was

22

performed by one-way ANOVA followed by Tukey’s post hoc test when F achieved P < 0.05, and

23

there was no significant variance in homogeneity. Some results were normalized to control to avoid

24

unwanted sources of variation. Statistical significance was set at P < 0.05.

25

■ ASSOCIATED CONTENT

26

SUPPORTING INFORMATION

27

UV, IR, MS, NMR, and HPLC spectras of (+)-1, (-)-1, (+)-2, and (-)-2, and partial results are given

28

in the Supplementary Information. Crystallographic data (CIF file) can be obtained free of charge

29

from the Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_request/cif.

with slight modifications. Briefly, at the last day of MWM test, the brains were quickly removed

17



1

■ AUTHOR INFORMATION

2

Corresponding Author

3

*

4

Author Contributions

5

Hao Wang*, Fei Xiong, Xiao-Long Hu, and Wen-Cai Ye conceived and designed the study.

6

Xiao-Long Hu, Cui Guo, Ji-Qin Hou performed the experiments. Jia-Hao Feng and Xiao-Qi Zhang

7

provided the technical assistance. Xiao-Long Hu and Cui Guo wrote the paper. All authors read and

8

approved the manuscript.

9

Notes

H. Wang: e-mail: [email protected]

10

The authors declare no competing financial interest.

11

■ ACKNOWLEDGMENTS

12

This work was financially supported by the National Natural Science Foundation of China (No.

13

81573309 and No. 81473160), and the Research and Innovation Project for College Graduates of

14

Jiangsu Province 2015 (No. KYLX15_0661), and Postgraduate Research & Practice Innovation

15

Program of Jiangsu Province (KYCX17_0694).

16

■ ABBREVIATIONS USED

17

GSK-3β, Glycogen synthase kinase-3β kinase; TCM, Traditional Chinese Medicine; Sch B,

18

Schisandrin B; AD, Alzheimer’s disease; FRET, Fluorescence resonance energy transfer; ELISA,

19

Enzyme-linked immunosorbent assay; NMR, Nuclear magnetic resonance; 1H-1H COSY, 1H-1H

20

orrelation spectroscopy; HSQC, Heteronuclear single quantum coherence; HMBC, Heteronuclear

21

multiple bond correlation.

22

■ REFERENCES

23

(1). aHooper, C., Killick, R., and Lovestone, S. (2008) The GSK3 hypothesis of Alzheimer's disease,

24

J Neurochem 104, 1433-1439.

25

(2). DaRocha-Souto, B., Coma, M., Perez-Nievas, B. G., Scotton, T. C., Siao, M., Sanchez-Ferrer,

26

P., Hashimoto, T., Fan, Z., Hudry, E., Barroeta, I., Sereno, L., Rodriguez, M., Sanchez, M. B.,

27

Hyman, B. T., and Gomez-Isla, T. (2012) Activation of glycogen synthase kinase-3 beta

28

mediates beta-amyloid induced neuritic damage in Alzheimer's disease, Neurobiol Dis 45,

29

425-437. 18


Page 18 of 39

Page 19 of 39 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1 2


(3). Maqbool, M., Mobashir, M., and Hoda, N. (2016) Pivotal role of glycogen synthase kinase-3: A therapeutic target for Alzheimer's disease, Eur J Med Chem 107, 63-81.

3

(4). Liang, S. H., Chen, J. M., Normandin, M. D., Chang, J. S., Chang, G. C., Taylor, C. K., Trapa,

4

P., Plummer, M. S., Para, K. S., Conn, E. L., Lopresti-Morrow, L., Lanyon, L. F., Cook, J. M.,

5

Richter, K. E. G., Nolan, C. E., Schachter, J. B., Janat, F., Che, Y., Shanmugasundaram, V.,

6

Lefker, B. A., Enerson, B. E., Livni, E., Wang, L., Guehl, N. J., Patnaik, D., Wagner, F. F.,

7

Perlis, R., Holson, E. B., Haggarty, S. J., El Fakhri, G., Kurumbail, R. G., and Vasdev, N. (2016)

8

Discovery of a Highly Selective Glycogen Synthase Kinase-3 Inhibitor (PF-04802367) That

9

Modulates Tau Phosphorylation in the Brain: Translation for PET Neuroimaging, Angew Chem

10 11 12

Int Edit 55, 9601-9605. (5). Sun, Z. K., Yang, H. Q., and Chen, S. D. (2013) Traditional Chinese medicine: a promising candidate for the treatment of Alzheimer's disease, Transl Neurodegener 2, 6.

13

(6). Zhao, H. H., Di, J., Liu, W. S., Liu, H. L., Lai, H., and Lu, Y. L. (2013) Involvement of GSK3

14

and PP2A in ginsenoside Rb1's attenuation of aluminum-induced tau hyperphosphorylation,

15

Behav Brain Res 241, 228-234.

16

(7). Park, H. J., Lee, K., Heo, H., Lee, M., Kim, J. W., Whang, W. W., Kwon, Y. K., and Kwon, H.

17

(2008) Effects of Polygala tenuifolia root extract on proliferation of neural stem cells in the

18

hippocampal CA1 region, Phytother Res 22, 1324-1329.

19

(8). Zhu, S., Wang, Y., Chen, M., Jin, J., Qiu, Y., Huang, M., and Huang, Z. (2012) Protective effect

20

of schisandrin B against cyclosporine A-induced nephrotoxicity in vitro and in vivo, Am J Chin

21

Med 40, 551-566.

22 23

(9). Song, L. R. State Administration of Traditional Chinese Medicine. Zhonghuabencao, Shanghai Science and Technology Publishing Company, Shanghai, 1999, 2902-2911.

24

(10). E, Q., Tang, M., Zhang, X., Shi, Y., Wang, D., Gu, Y., Li, S., Liang, X., Wang, Z., and Wang,

25

C. (2015) Protection of seven dibenzocyclooctadiene lignans from Schisandra chinensis against

26

serum and glucose deprivation injury in SH-SY5Y cells, Cell Biol Int 39, 1418-1424.

27

(11). Szopa, A., Ekiert, R., and Ekiert, H. (2017) Current knowledge of Schisandra chinensis (Turcz.)

28

Baill. (Chinese magnolia vine) as a medicinal plant species: a review on the bioactive

19



Page 20 of 39

1

components, pharmacological properties, analytical and biotechnological studies, Phytochem

2

Rev 16, 195-218.

3

(12). Nakajima, K., Taguchi, H., Ikeya, Y., Endo, T., and Yosioka, I. (1983) Constituents of

4

Schizandra chinensis Baill. XIII. Quantitative analysis of lignans in the fruits of Schizandra

5

chinensis Baill. by high performance liquid chromatography, Yakugaku Zasshi 103, 743-749.

6

(13). Giridharan, V. V., Thandavarayan, R. A., Arumugam, S., Mizuno, M., Nawa, H., Suzuki, K.,

7

Ko, K. M., Krishnamurthy, P., Watanabe, K., and Konishi, T. (2015) Schisandrin B Ameliorates

8

ICV-Infused Amyloid beta Induced Oxidative Stress and Neuronal Dysfunction through

9

Inhibiting RAGE/NF-kappa B/MAPK and Up-Regulating HSP/Beclin Expression, Plos One 10.

10

(14). Choi, Y. W., Takamatsu, S., Khan, S. I., Srinivas, P. V., Ferreira, D., Zhao, J. P., and Khan, I. A.

11

(2006)

12

Structure-antioxidant activity relationships of dibenzocyclooctadiene lignans, J Nat Prod 69,

13

356-359.

Schisandrene,

a

dibenzocyclooctadiene

lignan

from

Schisandra

chinensis:

14

(15). Guo, L. Y., Hung, T. M., Bae, K. H., Shin, E. M., Zhou, H. Y., Hong, Y. N., Kang, S. S., Kim,

15

H. P., and Kim, Y. S. (2008) Anti-inflammatory effects of schisandrin isolated from the fruit of

16

Schisandra chinensis Baill, Eur J Pharmacol 591, 293-299.

17

(16). Kim, S. R., Lee, M. K., Koo, K. A., Kim, S. H., Sung, S. H., Lee, N. G., Markelonis, G. J., Oh,

18

T. H., Yang, J. H., and Kim, Y. C. (2004) Dibenzocyclooctadiene lignans from Schisandra

19

chinensis protect primary cultures of rat cortical cells from glutamate-induced toxicity, J

20

Neurosci Res 76, 397-405.

21 22

(17). Liu, Z., Zhang, B., Liu, K., Ding, Z., and Hu, X. (2012) Schisandrin B attenuates cancer invasion and metastasis via inhibiting epithelial-mesenchymal transition, Plos One 7, e40480.

23

(18). Wei, B., Li, Q., Fan, R., Su, D., Ou, X., Chen, K., Chen, X., Jia, Y., and Bi, K. (2013)

24

UFLC-MS/MS method for simultaneous determination of six lignans of Schisandra chinensis

25

(Turcz.) Baill. in normal and insomniac rats brain microdialysates and homogenate samples:

26

towards an in-depth study for its sedative-hypnotic activity, J Mass Spectrom 48, 448-458.

27

(19). Li, X. J., Zhao, B. L., Liu, G. T., and Xin, W. J. (1990) Scavenging effects on active oxygen

28

radicals by schizandrins with different structures and configurations, Free Radic Biol Med 9,

29

99-104. 20


Page 21 of 39 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1 2 3 4


(20). Testa, B. (2015) Types of stereoselectivity in drug metabolism: a heuristic approach, Drug Metab Rev 47, 239-251. (21). Aboul-Enein HY, & Wainer IW (1997) The impact of stereochemistry on drug development and use. Wiley: New York.

5

(22). Rui, T., Lian-Niang, L., and Qicheng, F. (1984) Studies on the Chemical Constituents of

6

Kadsura longipedunculata: Isolation and Structure Elucidation of Five New Lignans, Planta

7

Med 50, 414-417.

8

(23). Ikeya, Y., Taguchi, H., Yosioka, I., and Kobayashi, H. (1979) Constituents of

9

Schizandra-Chinensis Baili .3. Structures of 4 New Lignans, Gomisin-H and Its Derivatives,

10

Angeloyl-Gomisin-H Tigloyl-Gomisin-H and Benzoyl-Gomisin-H, Chem Pharm Bull 27,

11

1576-1582.

12

(24). Khanfar, M. A., Hill, R. A., Kaddoumi, A., and El Sayed, K. A. (2010) Discovery of novel

13

GSK-3beta inhibitors with potent in vitro and in vivo activities and excellent brain permeability

14

using combined ligand- and structure-based virtual screening, J Med Chem 53, 8534-8545.

15

(25). Mudireddy, S. R., Abdul, A. R. M., Gorjala, P., and Gary, R. K. (2014) Beryllium is an

16

inhibitor of cellular GSK-3 beta that is 1,000-fold more potent than lithium, Biometals 27,

17

1203-1216.

18

(26). Arfeen, M., Patel, R., Khan, T., and Bharatam, P. V. (2015) Molecular dynamics simulation

19

studies of GSK-3beta ATP competitive inhibitors: understanding the factors contributing to

20

selectivity, J Biomol Struct Dyn 33, 2578-2593.

21 22

(27). Rester, U. (2008) From virtuality to reality - Virtual screening in lead discovery and lead optimization: a medicinal chemistry perspective, Curr Opin Drug Discov Devel 11, 559-568.

23

(28). Bertrand, J. A., Thieffine, S., Vulpetti, A., Cristiani, C., Valsasina, B., Knapp, S., Kalisz, H. M.,

24

and Flocco, M. (2003) Structural characterization of the GSK-3 beta active site using selective

25

and non-selective ATP-mimetic inhibitors, J Mol Biol 333, 393-407.

26 27 28 29

(29). Ravitz, M. J., and Wenner, C. E. (1997) Cyclin-dependent kinase regulation during G1 phase and cell cycle regulation by TGF-beta, Adv Cancer Res 71, 165-207. (30). Woodgett, J. R. (1990) Molecular-Cloning and Expression of Glycogen-Synthase Kinase-3 Factor-A, Embo J 9, 2431-2438. 21



1

(31). Pei, J. J., Tanaka, T., Tung, Y. C., Braak, E., Iqbal, K., and GrundkeIqbal, I. (1997) Distribution,

2

levels, and activity of glycogen synthase kinase-3 in the Alzheimer disease brain, J Neuropath

3

Exp Neur 56, 70-78.

4 5 6

(32). Leroy, K., and Brion, J. P. (1999) Developmental expression and localization of glycogen synthase kinase-3beta in rat brain, J Chem Neuroanat 16, 279-293. (33). Sereno, L., Coma, M., Rodriguez, M., Sanchez-Ferrer, P., Sanchez, M. B., Gich, I., Agullo, J.

7

M., Perez, M., Avila, J., Guardia-Laguarta, C., Clarimon, J., Lleo, A., and Gomez-Isla, T. (2009)

8

A novel GSK-3beta inhibitor reduces Alzheimer's pathology and rescues neuronal loss in vivo,

9

Neurobiol Dis 35, 359-367.

10

(34). Takashima, A. (2009) Drug development targeting the glycogen synthase kinase-3beta

11

(GSK-3beta)-mediated signal transduction pathway: role of GSK-3beta in adult brain, J

12

Pharmacol Sci 109, 174-178.

13

(35). Kozikowski, A. P., Gaisina, I. N., Petukhov, P. A., Sridhar, J., King, L. T., Blond, S. Y., Duka,

14

T., Rusnak, M., and Sidhu, A. (2006) Highly potent and specific GSK-3 beta inhibitors that

15

block Tau phosphorylation and decrease alpha-synuclein protein expression in a cellular model

16

of Parkinson's disease, Chemmedchem 1, 256-266.

17

(36). Khanfar, M. A., Asal, B. A., Mudit, M., Kaddoumi, A., and El Sayed, K. A. (2009) The marine

18

natural-derived inhibitors of glycogen synthase kinase-3beta phenylmethylene hydantoins: In

19

vitro and in vivo activities and pharmacophore modeling, Bioorg Med Chem 17, 6032-6039.

20

(37). Wagman, A. S., Johnson, K. W., and Bussiere, D. E. (2004) Discovery and development of

21

GSK3 inhibitors for the treatment of type 2 diabetes, Curr Pharm Des 10, 1105-1137.

22

(38). Ma, Q. L., Lim, G. P., Harris-White, M. E., Yang, F., Ambegaokar, S. S., Ubeda, O. J., Glabe,

23

C. G., Teter, B., Frautschy, S. A., and Cole, G. M. (2006) Antibodies against beta-amyloid

24

reduce Abeta oligomers, glycogen synthase kinase-3beta activation and tau phosphorylation in

25

vivo and in vitro, J Neurosci Res 83, 374-384.

26

(39). Garcia-Alloza, M., Robbins, E. M., Zhang-Nunes, S. X., Purcell, S. M., Betensky, R. A., Raju,

27

S., Prada, C., Greenberg, S. M., Bacskai, B. J., and Frosch, M. P. (2006) Characterization of

28

amyloid deposition in the APPswe/PS1dE9 mouse model of Alzheimer disease, Neurobiology

29

of Disease 24, 516-524. 22


Page 22 of 39

Page 23 of 39 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

(40). Elagawany, M., Ibrahim, M. A., Ahmed, H. E. A., El-Etrawy, A. S., Ghiaty, A., Abdel-Samii, Z.

2

K., El-Feky, S. A., and Bajorath, J. (2013) Design, synthesis, and molecular modelling of

3

pyridazinone and phthalazinone derivatives as protein kinases inhibitors, Bioorg Med Chem Lett

4

23, 2007-2013.

5

(41). Lee, M. R., Yun, B. S., Park, S. Y., Ly, S. Y., Kim, S. N., Han, B. H., and Sung, C. K. (2010)

6

Anti-amnesic effect of Chong-Myung-Tang on scopolamine-induced memory impairments in

7

mice, J Ethnopharmacol 132, 70-74.

8

(42). Zhao, H. F., Li, N., Wang, Q., Cheng, X. J., Li, X. M., and Liu, T. T. (2015) Resveratrol

9

decreases the insoluble Abeta1-42 level in hippocampus and protects the integrity of the

10

blood-brain barrier in AD rats, Neuroscience 310, 641-649.

11

23



1 2

Figure 1

3

24


Page 24 of 39

Page 25 of 39 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1 2

Figure 2

3 4

Figure 3

25



1 2

Figure 4

26


Page 26 of 39

Page 27 of 39 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1 2

Figure 5

27



1 2

Figure 6

28


Page 28 of 39

Page 29 of 39 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1 2

Figure 7

3

29



1

Page 30 of 39

Table 1 The inhibitory activities of target compounds against GSK-3β Compounds (+)-1 (-)-1 (+)-2 (-)-2 Bioc

GSK-3β inhibition (10 µM) 89.23 87.34 90.23 92.12 -

Docking scoreb (cal/mol) -12.34 -14.58 -39.45 -42.12 -5.32

IC50 (nM)a 341.32 ± 12.3 290.89 ± 15.7 80.67 ± 5.5 73.22 ± 4.2 25.49 ± 3.11

2

a

All IC50 values were the mean of at least three independent determinations.

3

b

Docking scores were obtained from Discovery studio (2.5) software.

4

c

Bio was used as a reference compound

5

Table 2 The inhibitory activities of test compounds on other homologous proteins

IC50 values (µM) Protein Kinases (+)-1

(-)-1

(+)-2

(-)-2

PI4KB

> 100

> 100

> 100

> 100

DYRK1A

> 100

> 100

> 100

> 100

DYRK1B

> 100

> 100

> 100

> 100

MAP4K4

> 100

> 100

> 100

> 100

MNK1

> 100

> 100

> 100

> 100

CDK1/cyclinB

89.00

92.23

83.23

80.44

CDK2/cyclinA

50.00

56.34

60.43

66.00

CDK5/p25

> 100

> 100

> 100

> 100

CDK5/p35

> 100

> 100

> 100

> 100

CDK9/cyclinT1

> 100

> 100

> 100

> 100

GSK-3β

0.34

0.29

0.08

0.07

GSK-3α

5.80

3.12

1.65

2.11

6 7 8 9 10 30


Page 31 of 39 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

Figure legends:

2

Figure 1 Chemical structures of (+)-1, (-)-1, (+)-2, and (-)-2.

3

Figure 2. X-ray ORTEP drawings of (+)-1, (-)-1, (+)-2, and (-)-2.

4

Figure 3. 2D and 3D docked binding modes of compounds-GSK-3β complexes. The dotted line

5

represents H-interaction, and the red dotted line represents π-interaction.

6

Figure 4. Kinetic data determined for compound (+)-1, (-)-1, (+)-2, and (-)-2. (A) ATP concentrations

7

varied from 0.5 to 8 µM. Compound concentrations used were depicted in the plot, and GS-2

8

concentration was kept constant at 6.25 µM. (B) GS-2 concentrations varied from 0.78 to 12.5 µM.

9

Compound concentrations used were depicted in the plot, and ATP concentration was kept constant

10

at 2 µM. Each point was the mean of triplicate determinations.

11

Figure 5. Effects of (+)-1, (-)-1, (+)-2, and (-)-2 on the expression of p-GSK-3β, p-tau, and on the

12

level of liver glycogen in vivo. (A) Immunohistochemistry analyses of p-GSK-3β and p-tau in

13

hippocampus. (B) Fluorescent quantitative analysis of p-GSK-3β (Ser9). (C) Fluorescent quantitative

14

analysis of p-Tau (Thr231). (D) Western blot analyses of p-GSK-3β and p-tau expressions in

15

hippocampus, using total proteins as control. (E) The level of liver glycogen. Data are presented as

16

Means ± S.E.M. (n=10 mice/group). *p < 0.05, **p < 0.01, and ***p < 0.001 compared to control

17

group.

18

Figure 7. The effects of (±)-1 and (±)-2 on cognitive impairments, Aβ1-42 level, and p-GSK-3β/tau

19

pathways (n=10 mice/group). (A) Schematic of experimental procedure. (B) Acquisition trial. (C)

20

Probe trial of Morris water maze in APP/PS1 mice. (D)The times across platform. (E) Representative

21

searching swimming paths by mice with different treatments in the probe trial test. (F) The Aβ1-42

22

level in hippocampus. (G) Western-blot analysis of p-GSK-3β and p-tau in hippocampus of APP/PS1

23

mice. **p < 0.05 compared to WT mice, ##P < 0.05 compared to vehicle-treated APP/PS1 mice.

24

31



Figure 1 Chemical structures of (+)-1, (-)-1, (+)-2, and (-)-2. 166x146mm (300 x 300 DPI)


Page 32 of 39

Page 33 of 39 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 2. X-ray ORTEP drawings of (+)-1, (-)-1, (+)-2, and (-)-2. 174x161mm (300 x 300 DPI)



Figure 3. 2D and 3D docked binding modes of compounds-GSK-3β complexes. The dotted line represents Hinteraction, and the red dotted line represents π-interaction. 117x73mm (300 x 300 DPI)


Page 34 of 39

Page 35 of 39 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 4. Kinetic data determined for compound (+)-1, (-)-1, (+)-2, and (-)-2. (A) ATP concentrations varied from 0.5 to 8 µM. Compound concentrations used were depicted in the plot, and GS-2 concentration was kept constant at 6.25 µM. (B) GS-2 concentrations varied from 0.78 to 12.5 µM. Compound concentrations used were depicted in the plot, and ATP concentration was kept constant at 2 µM. Each point was the mean of triplicate determinations. 67x23mm (300 x 300 DPI)



Figure 5. Effects of (+)-1, (-)-1, (+)-2, and (-)-2 on the expression of p-GSK-3β, p-tau, and on the level of liver glycogen in vivo. (A) Immunohistochemistry analyses of p-GSK-3β and p-tau in hippocampus. (B) Fluorescent quantitative analysis of p-GSK-3β (Ser9). (C) Fluorescent quantitative analysis of p-Tau (Thr231). (D) Western blot analyses of p-GSK-3β and p-tau expressions in hippocampus, using total proteins as control. (E) The level of liver glycogen. Data are presented as Means ± S.E.M. (n=10 mice/group). *p < 0.05, **p < 0.01, and ***p < 0.001 compared to control group. 206x224mm (300 x 300 DPI)


Page 36 of 39

Page 37 of 39 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 6. Effects of (+)-1, (-)-1, (+)-2, and (-)-2 on cell viability, and the expression of p-GSK-3β and the expression of p-tau in SH-SY5Y cells. (A) Cell viability assay. (B) Western blot analyses of the Bcl-2 and Bax expressions, using β-actin as control. (C) Western blot analyses of the p-GSK-3β and p-tau expressions, using total proteins as control. (D) The immunocytochemistry analyses of p-GSK-3β and p-tau in SH-SY5Y cells. (E) Fluorescent quantitative analysis of p-GSK-3β (Ser9). (F) Fluorescent quantitative analysis of pTau (Thr231). Data are presented as Means ± S.E.M. (n=5). *p < 0.05, **p < 0.01 or ***p < 0.001compared to control group. #P< 0.05, ##P < 0.01 or ###P < 0.001 compared to Aβ-treated group. 217x248mm (300 x 300 DPI)



Figure 7. The effects of (±)-1 and (±)-2 on cognitive impairments, Aβ1-42 level, and p-GSK-3β/tau pathways (n=10 mice/group). (A) Schematic of experimental procedure. (B) Acquisition trial. (C) Probe trial of Morris water maze in APP/PS1 mice. (D)The times across platform. (E) Representative searching swimming paths by mice with different treatments in the probe trial test. (F) The Aβ1-42 level in hippocampus. (G) Western-blot analysis of p-GSK-3β and p-tau in hippocampus of APP/PS1 mice. **p < 0.05 compared to WT mice, ##P < 0.05 compared to vehicle-treated APP/PS1 mice. 186x183mm (300 x 300 DPI)


Page 38 of 39

Page 39 of 39 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Table of contents 45x23mm (300 x 300 DPI)


Stereoisomers of Schisandrin B Are Potent ATP ... - ACS Publications

Recommend Documents