The Synthesis and Characterization of AuNCs
A typical NIBC molecule modified AuNCs with a diameter lower than 2 nm was synthesized in one-pot by using a two-step method according to our previous works26, 27, 30. Firstly, the N-isobutyryl-L-cysteine (NIBC) and HAuCl4 were added into a cooled methanol solution at 0 °C (cool bath). Then the mixtures were incubated in cool bath for 1 h with a gently stirring to get a react intermediates of Au+(NIBC) complex. Secondly, NaBH4 was added into the mixtures and incubated for another 0.5 h with a strongly stirring in room temperature to get the final products of AuNCs. After dialysis and lyophilization, the pure AuNCs were characterized by the TEM, UV-vis-NIR absorption spectroscopy, XPS and FT-IR spectroscopy.
Investigation of the interaction between olanzapine and AuNCs via UV-Vis Titration
UV-vis titration experiments were adopted to investigate the interaction between olanzapine molecules and AuNCs. UV-vis titration measurements were carried out in a Shimadzu UV-1800 UV-vis spectrometer with a 1 cm path-length quartz cuvette. The host samples solutions of AuNCs in dimethyl sulfoxide (DMSO) with a concentration of 500 μg/mL were prepared at room temperature (about 25 °C). The guest solution of 10000 μg/mL olanzapine in DMSO were also prepared at room temperature. The mother liquor of 3.0 mL of AuNCs solution were titrated by successive additions of fixed small aliquots (10 μL) of the guest olanzapine solution. Titrations were done manually by using trace syringes, and the UV-vis intensity was measured after every drops. The UV-vis adsorption peak of AuNCs in DMSO was also at 670 nm, same to that in water. 36 times of titrations were carried out in this study. In order to exclude the decrease of UV-vis peak intensity caused by solvent dilution, a blank control group was adopted by using DMSO solvent as the guest solution. The mother liquor of 3.0 mL of AuNCs solution were titrated by successive additions of fixed small aliquots (10 μL) of DMSO.
Cell line and olanzapine powder
SH-SY5Y cell line was purchased from Bluef (Shanghai) Biotechnology Development Co., Ltd, Shanghai, China. The SH-SY5Y were cultured in Dulbecco's Modified Eagle Medium /Nutrient Mixture F-12 (DMEM/F12) (BL305A, Lanjike Technology Co., Ltd, Anhui, China), 15-20% fetal bovine serum (11011-8611, Zhejiang Tianhang Biological Technology Co., Ltd, Zhejiang, China) and 1% penicillin/streptomycin (A3160801 and 15140-122, Thermo Fisher Scientific (China) Co., Ltd, Shanghai, China), at 37 °C in a humidified 5% CO2 incubator. Olanzapine powder was purchased from Sigma (LRAC0272) and was dissolved in Dimethyl sulfoxide (DMSO). Control cells were treated with DMEM + DMSO (vehicle). Cells were treated for 2 h and 24 h.
The effect of AuNCs on olanzapine-induced H1R-AMPK signal dysfunction in vitro
The effect of olanzapine and AuNCs on H1R and pAMPK signaling in cells were examined by immunostaining. In brief, SH-SY5Y cells were plated in 6-well plates on a glass coverslip coated with poly-d-lysine, grown at the corresponding temperatures (37 °C in a humidified 5% CO2). Cells were divided into five groups (n = 4/group) and treated with vehicle, olanzapine-50 μM, olanzapine-50 μM + AuNCs-20 mg/L, olanzapine-50 μM + AuNCs-10 mg/L, AuNCs-20 mg/L for 2 h and 24 h. Cells were fixed with 4% paraformaldehyde (PFA), washed with PBS, repaired with EDTA antigen retrieval buffer (Beijing Solarbio Science & Technology, C1034) and blocked with 10% BSA. Then, cells were incubated with primary antibodies (H1R and pAMPK (bs-6663R, bsm-52132R, Beijing Biosynthesis Biotechnology). The cells were incubated with goat anti-rabbit secondary antibodies (ab6717, Abcam Trading; GB21303, Wuhan Google Biotechnology). The nucleus was counterstained by DAPI (C0065, Beijing Solarbio Technology) for 10 min at room temperature in the dark. The percentage area of the H1R and pAMPK over the total area was measured by the software Image-pro plus 6.0 (Media Cybernetics, Inc., Rockville, MD, USA).
Animals and Olanzapine tablets
Female Sprague Dawley (SD) rats (9-10 weeks) were obtained from the Animal resources center (SPF (Beijing) Biotechnology Co., Ltd, Beijing, China). Female C57BL/6 and BALB/c mice (weight 18-20 g) were purchased from Hubei experimental animal research center and Zhejiang weitong lihua experimental animal technology Co. Ltd., China. Animals were housed in barrier system (22 ± 2 °C on a 12 h light-dark cycle, lights on 07:00). All the animals were fed a standard diet throughout the studies. Olanzapine tablets was purchased from Eli Lilly, S.A. Madrid, Spain. The animal experiments were approved by the Animal Ethics Committee of Wuhan University of Technology (SYXK 2017-0092), and carried out according to the institutional guidelines. All experiments performed were following relevant guidelines and regulations. In addition, all experiments were carried out in compliance with the ARRIVE guidelines.
The preventive effect of AuNCs on olanzapine-induced weight gain in rats
Rats were divided into 5 groups randomly (n = 10-12/group): control (CON, group1), olanzapine-only (OLZ, group 2), co-treatment of olanzapine and AuNCs high dose (OLZ + AuNCs H, group 3), co-treatment of olanzapine and AuNCs low dose (OLZ + AuNCs L, group 4), AuNCs high dose-only (AuNCs H, group 5). Olanzapine was mixed with 0.3 g cookie-dough pellet according to our previous protocol36. From the beginning of the experiment, rats in group 2-4 received oral olanzapine administration (1 mg/kg, t.i.d, at 07:00, 15:00 and 23:00). Rats in group 1 and group 5 were given vehicle (cookie with no olanzapine) as control. From the first day of olanzapine (or vehicle) administration, rats in OLZ + AuNCs H and OLZ + AuNCs L groups received 20 mg/kg or 10 mg/kg AuNCs treatment through IP injection (once a day). Rats in CON and OLZ group received an IP injection of saline. Rats in AuNCs high dose-only group were treated with 20 mg/kg AuNCs as a drug control group. The above treatment was last for 21 days. Food intake was measured every 24 h and weight gain was measured every 48 h. After the last treatment, rats were euthanized by fast CO2 infusion32. The plasma was collected and stored in -80℃. BAT and hypothalamus were dissected stored in -80°C. Half of the BAT tissues were fixed in 4% PFA and used for immunohistochemistry analysis.
The therapeutical effect of AuNCs on established obesity induced by olanzapine
Rats were divided into two groups including olanzapine (OLZ, n = 33) and control (CON, n = 22) groups. The rats in CON group were treated with vehicle, and rats in OLZ group were treated with olanzapine the same as in Animal experiment 1 for 28 days. From day 29, olanzapine treated rats were randomly divided in to 3 groups including olanzapine only (OLZ), olanzapine + AuNCs high dose (OLZ + AuNCs H, 20 mg/kg), olanzapine + AuNCs low dose (OLZ + AuNCs L, 10 mg/kg) groups and were treated with AuNCs and olanzapine based on grouping for 28 days. The rats treated with vehicle were randomly divided into 2 groups including control (CON) group and AuNCs high dose (AuNCs H, 20 mg/kg, IP) group. Rats in CON group were IP injected with saline and rats in AuNCs H were IP injected with 20 mg/kg AuNCs for 28 days. The body weight and food intake of each rat was measured every 48 h and 24 h, respectively.
Investigation of the toxicity of AuNCs during short-term and long-term treatment
In short-term treatment, 15 female mice were randomly divided into 3 groups: control, AuNCs 40 mg/kg and AuNCs 160 mg/kg (n = 5/group). The mice received AuNCs or saline treatment by IP injection for 28 days (once a day). Mouse behaviors were observed every day. After the last treatment, mice were sacrificed and the blood were collected. The biochemistry markers including TBIL, TP, ALB, ALT, UREA, CREA, GLU and CHOL were examined by using a biochemical analyzer. In long-term treatment, 15 female mice were randomly divided into control group and AuNCs group. The mice received a therapeutical dose of AuNCs (20 mg/kg) or saline treatment by IP injection (once a day) for 6 months. After the last treatment, mice were sacrificed and tissues including brain, heart, liver, spleen, lung, and kidney were collected and used for histology.
Glucose intolerance test
Rats were fasted for 16 h with free of water. Blood was collected from the tail vein of rats, and a fasting blood glucose (0 min) was measured using a blood glucose meter (Johnson & Johnson one touch ultra, Johnson & Johnson (China) Medical Equipment Co., Ltd.). Then rats were IP injected with 1 g/kg glucose. The blood glucose at 30th min, 60th min, 90th min and 120th min post injection was detected. The area under curve (AUC) of glucose level was calculated for each rat.
Plasma Hormone Analysis and Biochemical Analysis
Levels of leptin, insulin, cholesterol and triglycerides in the plasma were analyzed by commercially available ELISA Kits (leptin, Elabscience Biotechnology Co., Ltd., Hubei, China, E-EL-R0582c; insulin, E-EL-R2466c, cholesterol, Changchun Huili Biotechnology Co., Ltd., Jilin, China, C048-a; triglycerides, C019-a). The absorbance of the samples was measured with a FlexStation 3 Multi-Mode Microplate Reader (Molecular Devices).
Open Filed Test
Rats were subjected to open field behavioral testing to examine the locomotor activity of each rat. In brief, rats were put into an open field experiment box (60 cm × 60 cm × 40 cm). A high-frequency camera was used to track and record the behavior changes of rats within 30 min on free movement in the open field equipment. Total distance travelled (m) were recorded. Feces of each rat was cleaned after the experiment, and the bottom surface was cleaned with 75% ethanol solution and wiped to prevent odor infection.
Histology (H&E staining)
To access the morphology of the BAT adipocytes in olanzapine and AuNCs treated rats, the PFA-embedded BAT tissue was section-sliced (4 μm/section) and mounted on glass slides (n = 4/group). Two fields per section were randomly captured with a Nikon camera (Eclipse ci, Nikon Instruments (Shanghai) Co., Ltd., Shanghai, China) at 10× objective. The percentage area of the multilocular brown adipocytes over the total area was measured by the software Image-pro plus 6.0 (Media Cybernetics, Inc., Rockville, MD, USA). To investigate the toxicity of AuNCs on mice, PFA- embedded tissues were sectioned by Leica pathology microtome (4 μm/section) and treated with hematoxylin and eosin-staining immediately (n = 3/group). The histological sections were examined by biological microscope (Olympus) with 20× objective lens.
Western Blot Procedures
The protein expression of key molecules of the H1R-AMPK signaling, POMC and UCP-1 signaling were examined by western blot. In brief, tissues were homogenized and the protein concentration was determined by BCA protein concentration assay. Protein was separated and then transferred onto polyvinylidene difluoride membranes and incubated with primary antibodies (H1R, Wuhan Sanying Biotechnology, 13413-1-ap; AMPK, Affinity, Df6361; pAMPK, Affinity, Af3423; POMC, Wuhan Sanying Biotechnology, 66358-1-ig; UCP-1, Wuhan Sanying Biotechnology, 23673-1-ap; PGC-1-α, Wuhan Sanying Biotechnology, 20658-1-ap; PPAR-α, Wuhan Sanying Biotechnology, 15540-1-ap; PPAR-γ, Wuhan Sanying Biotechnology, 16643-1-AP) and secondary antibodies (BOSTER Biological Technology, BA1051 and BA1054). The enhanced chemiluminescence (ECL) kit were used to analyze the band. The quantification of protein was normalized to those of β-actin (BOSTER Biological Technology, BM0627).
Immunohistochemistry
Immunohistochemistry was used to examined the expression of UCP-1and PGC-1 in BAT according to the previous work26. BAT sections were incubated with 3% hydrogen peroxide solution in the dark at room temperature, and were blocked with 3% BSA. The sections were then incubated with the primary antibodies and secondary antibodies (UCP-1, Wuhan servicebio technology, GB11370-1; PGC-1α, Wuhan servicebio technology, GB14097). The sections were then counterstained with haematoxylin. The UCP-1 and PGC-1α were counted by ratio of positive cells to total tissue area in each field by the software Image-pro plus 6.0 (Media Cybernetics, Inc., Rockville, MD, USA).
Statistical Analysis
SPSS 22.0 (IBM SPSS Statistics, USA) was used for statistical analysis. The distributions of data were detected by Kolmogorov-Smirnov test. The statistical differences of normal distributed data were analyzed by one-way ANOVA with followed by post hoc Dunnett t-test for multiple comparisons. Pearson’s correlation test was used to analyze the relationships among the measurements. The data was expressed as Mean ± SEM. p < 0.05 was considered statistically significant.