BDNF-ERK-CREB Signaling Mediates the Role of Mir-132 in the Regulation of the Effects Of

Antidepressant-like effects of standardized gypenosides: Involvement of brain-derived

neurotrophic factor signaling in hippocampus

Rong-Hao Mua, Xiao-Yan Fangb, Shuang-Shuang Wanga, Cheng-Fu Lic, Shao-Mei Chenc,

Xue-Mei Chenc, Qing Liu a,d, e,*, Yu-Cheng Lib, Li-Tao Yia,d,e,* a Department of Chemical and Pharmaceutical Engineering, College of Chemical

Engineering, Huaqiao University, Xiamen 361021, Fujian province, PR China b College of Pharmacy, Henan University of Traditional Chinese Medicine, Zhengzhou

450008, Henan province, PR China c Xiamen Hospital of Traditional Chinese Medicine, Xiamen 361009, Fujian province, PR

China d Institute of Pharmaceutical Engineering, Huaqiao University, Xiamen 361021, Fujian province, PR China e Fujian Provincial Key Laboratory of Biochemical Technology, Huaqiao University, Xiamen

361021, Fujian province, PR China

*Corresponding authors:

Dr. Li-Tao Yi. Tel./Fax.: 86-592-6162302; E-mail address: [email protected]; [email protected]

Dr. Qing Liu. Tel./Fax.: 86-592-6162302; E-mail address: [email protected]

The project was supported by grants from the National Natural Science Foundation of China

(No. 81202940; 81303278) and the Science Research Foundation of ministry of Health &

United Fujian Provincial Health and Education Project for Tacking the Key Research (WKJ-

FJ-31). The authors reported no biomedical financial interests or potential conflicts of interest.

1 Materials and methods

Open-field test

To assess the effect of gypenosides on locomotor activity, mice were administered with gypenosides (25, 50, 100 mg/kg) and then were evaluated in the open-field test 1 h later. The apparatus consisted of a wooden box measuring 40×40×30 cm, with the floor divided into 25 equal squares (8 × 8 cm). The numbers of squares crossed with all paws (crossing) and of standing on the hind legs (rearing) were counted in a 3-min session. The apparatus was cleaned with a detergent and dried after occupancy by each mouse.

Validation of BDNF antibody

To assess the validation of BDNF antibody, BDNF recombinant protein (Boster, China) was dissolved in PBS. Four different concentrations of BDNF were set in the validated experiment: the BDNF-25nmol group, the BDNF-50nmol group, the BDNF-100nmol group, the vehicle group. Then the BDNF bands were got in western blotting. The data were shown below (provided as Fig.1S). With the increase of BDNF concentration, there was a corresponding increase of BDNF band value in western blotting. Therefore, the BDNF antibody (Santa Cruz) and the data about BDNF expression in the text of manuscript are

2 reliable.

Fig.1S BDNF expression induced by different BDNF treatment.

Results

The effects of gypenosides on the open-field test in mice

As shown in the Fig.2S, one-way ANOVA revealed neither fluoxetine nor gypenosides showed a significant alteration on the crossing number [F(4,35)=1.003, P=0.419] or rearing number [F(4,35)=0.826, P=0.518] of open-field test in mice.

A B 80 30 r r e e

b 60 b

m 20 m u u n

g 40 g n i n i s r s

a 10 o e

r 20 R C

0 0 e u 5 0 0 e u 5 0 0 l l 2 5 0 l l 2 5 0 ic F - - 1 ic F - - 1 h s s - h s s - e e e s e e e s V id id e V id id e s s id s s id o o s o o s n n o n n o e e n e e n p p e p p e y y p y y p G G y G G y G G

Fig.2S Effects of gypenosides on the crossing (A) and rearing (B) numbers of mice in open-

3 field test (n=8).