Depression is a major mood disorder. Abnormal expression of glial glutamate transporter-1 (GLT-1) is associated with depression. Schisantherin B (STB) is one bioactive of lignans isolated from
Depression is regarded as a major mood disorder, characterized by either the altered mood or cognitive functions (Dillon and Pizzagalli, 2018). Late life depression, characterized by a pervasive and persistent low mood in elderly people, is invariably accompanied by numbers of cognitive impairments, including executive function, memory, processing speed, attention and visuospatial skill domains (Jeon and Kim, 2017; Liao
GLT-1 is termed as the major Na+-driven glutamate transporter. Maladaptation of GLT-1 has been recommended as a contributor to several neuropathological conditions related to neuro (Rodriguez-Kern
The PI3K pathway is known for regulating metabolism, together with cell growth and cell survival. Akt is one of the key molecules activated downstream of the PI3K. This kinase has been shown to play multiple roles in the cellular regulation and has emerged as a critical mediator of mammalian target of rapamycin (mTOR) activity (Sheppard
Experiment was carried out in compliance with the National Institutes of Health and institutional guidelines for the humane care of animals. Experimental procedure was approved by the Animal Care Committee of Shenyang Pharmaceutical University.
Male KM mice (The joint venture of Charles River Laboratories in China) were used in experiments. The mice at 10-week old (20–25 g) and 11-month old (50–60 g) were maintained on a 12-h light/dark cycle (lights on 07:00 to 19:00 h) with
STB was purchased from The National Institute for the Control of Pharmaceutical and Biological Products in China (Shenyang, China), with purity above 98%. Before the experiment, we used three mice per group to screen the dose of STB (10, 15 and 20 mg/kg) through FST. It was found that the STB of 15 mg/kg had better anti-depression effect in mice. Thus, the suspension solution was diluted with physiological saline to 1.5 mg/mL. Citalopram as the positive control drug was obtained from Melone Pharmaceutical Co (Dalian, China). It was dissolved in physiological saline at a concentration of 1 mg/mL. All other chemicals and reagents were of analytical grade.
Following 7 days of acclimation to the vivarium and housing conditions, mice were divided into 8 experimental groups shown as below, and 15 in each group, the experimental operation were parallel:
(1) Normal group-I (non-FST treated, 10-week old, n=15), (2) Control group-I (physiological saline, FST treated, 10-week old, n=15), (3) Citalopram group-I (FST treated; 10-week old, n=15), (4) STB group-I (FST treated; 10-week old, n=15), (5) Normal group-II (FST treated, 11-month old, n=15), (6) Control group-II (physiological saline, FST treated, 11-month old, n=15), (7) Citalopram group-II (FST treated; 11-month old, n=15) (8) STB group-II (FST treated, 11-month old, n=15). Citalopram (10 mg/kg, i.p.) and STB (15 mg/kg, i.p.) were administered daily between 09:00 and 10:00 h for 10 days. Except for the Normal group, FST was carried out on day 19. 30 min after FST, Y-maze test was implemented to assess immediate spatial working memory of animals. On the following day and the 27th day, Y-maze test was carried out again. 30 min before Y-maze test, OFT was carried out. After each Y-maze test, 5 mice in each group were sacrificed by cervical dislocation and the brain was immediately removed. The tissues were stored at −80°C. Brief experimental design is explained in Fig. 2.
Mice were subjected to the FST as described previously (Rojas
Y-maze test was carried out to assess immediate spatial working memory which is a form of short-term memory (Cognato Gde
OFT was conducted in a camera obscura (30 cm long×30 cm wide×30 cm high) which was made of black plexiglas and with digital auto photometer to detect the depressive-like response of mice (Xie
Hippocampal tissues were homogenized in RIPA (150 mM sodium chloride, 50 mM Tris (pH 8.0), 0.5% sodium deoxycholate, 0.1% SDS, 1% Triton X-100) and PMSF (Dalian Melonepharma, Dalian, China) and kept on ice for 30 min. The tissue homogenate was centrifuged at 10 000 g for 20 min at 4°C. The supernatant was obtained and used as the total hippocampal protein extract measured by BCA assay kit (Dalian Melonepharma) to determine protein concentration and stored at −80°C until use. Samples were diluted with an equal volume of loading buffer (Beyotime Biotech Co., Shanghai, China), and boiled at 95°C for 5 min. Approximately 50 μg of protein was loaded in each well and separated in 10% or 12% SDS-PAGE gels. The proteins were transferred onto nitrocellulose membranes. The membranes were saturated and blocked with 5% fat-free powdered milk at 37°C for 1.5 h and incubated overnight at 4°C in one of the following primary antibodies, which were diluted in 5% fat-free powdered milk in TBS: GLT-1 (1:1000, Abcam, San Francisco, CA, USA), PI3 Kinase p85 (19H8) (1:1000, CST, USA), Akt (pan) (C67E7) (1:1000, CST, USA), mTOR (7C10) (1:1000, CST, USA), β-actin Rabbit mAb (1:1000, CST, USA). Anti-PI3K p85 (phosphor Y607) (1:1000, Abcam), Phospho-Akt (Ser473) (1:2000, CST, USA), Phospho-mTOR (Ser2448) (1:1000, CST, USA). Blots were washed three times for 30 min in TBST at room temperature and then incubated for 1.5 h in one of the following HRP-conjugated antibodies which were diluted in 5% fat-free powdered milk in TBS: Anti-rabbit IgG (1:2000, CST, USA) for detection of target proteins, β-actin. After three times washes for 30 min in TBST, immunolabeled protein bands were detected using the ECL western blot detection kit (Dalian Melonepharma). Graphs of blots were obtained in the linear range of detection and were quantified for the level of specific induction by scanning laser densitometry.
Results are expressed as mean ± SEM. The significances between different groups were assessed using one-way ANOVA, followed by Tukey HSD post-hoc test when significant main effects were indicated. In all calculations, *
Fig. 3 showed the duration of immobility times (A), swimming times (B) and climbing times (C) measured during the 4 min test session. Indeed, whether for 10-week or 11-month old mice, compared with the control group, 10 days treatments of Citalopram (10 mg/kg; i.p.) or STB (15 mg/kg; i.p.) could significantly increase swimming time, reduce immobility time and climbing time (
To detect whether FST lead to a certain degree of cognitive impairment, we performed a Y-maze test after 30 min of FST and on day 1 (20th day of the total experiment) and day 7 (27th day of the total experiment) after FST, respectively. On the first test, we found that stress induced by FST significantly decreased the spontaneous alternation behavior of mice compared with normal group whether for 10-week or 11-month old mice, which indicated FST could impair short-term memory of mice, and Citalopram or STB treatment could protect mice from FST for 10-week old mice (
On the second time of Y-maze, the short-term learning and memory of control group had been restored to that of the nor- mal group for 10-week old mice (
To further study how many days the impact of FST on learning and memory could last, we carried out the Y-maze test again on the 27th day. As shown in Fig. 4C, there was no difference between all groups, which meant impairment from FST could restore to normal within 7 days.
As shown in Fig. 5A, on the 20th day, FST procedure resulted in significantly reduced total distance travelled and time in the central area in the OFT as compared to normal group for 11-month old mice (
In the 27th day, as shown in Fig. 5B, we found no difference in total track and time in the marginal area among all groups of mice (Fig. 5B-1, Fig. 5B-3), which meant the depressive-like behavior in mice induced by FST could last more than one day for the older mice. However, we still found a significant decrease of control group in the time in the central area compared with normal group, and there was no significant recall after STB or Citalopram administration (
In order to research the molecular changes in the brain, western blot was used to detect the effects of FST and STB treatment on GLT-1/PI3K/AKT/mTOR signaling pathways in the 19th, 20th, 27th day. From the results of the 19th day, compared with the control group, the FST procedure could significantly decrease the levels of GLT-1, P-PI3K/PI3K, P-AKT/AKT and P-mTOR/mTOR in hippocampus (
STB treatment could restore learning and memory more rapidly for 11-month old mice in 20th day (
Glutamate-related therapies are emerging as the new path for the treatment of major depressive disorder (MDD) (Miyagishi
Previous studies proposed that pharmacological up-regulation of GLT-1 could also ameliorate the age-dependent pathological tau accumulation (de Calignon
In summary, our research shows that the STB acting as an antidepressant increases GLT-1 levels by promoting PI3K/AKT/mTOR pathway, and also exert cognition improvement ability in the meanwhile. Our findings suggest that STB might be a promising therapeutic agent of depression, and further research is worth to be invested.
This research was supported by National Natural Science Foundation of China (No. 81573580) and Natural Science Foundation of Liaoning Province of China (No. 2014020076), Key Laboratory of Polysaccharide Bioactivity Evaluation of TCM of Liaoning Province and Key Laboratory of Quality Control of TCM of Liaoning Province (17-137-1-00).
The authors have no conflict of interest to declare.