Protective effects of luteolin on cognitive impairments induced by psychological stress in mice

Agents and chemicals

Luteolin (purity >98%) was purchased from Shanxi Sciphar Biotechnology Co. Ltd (Shanxi, China). Corticosterone, norepinephrine and dopamine enzyme-linked immunosorbent assay (ELISA) kits were purchased from Adlitteram Diagnostic Laboratories (San Diego, CA, USA). Malondialdehyde (MDA), superoxide dismutase (SOD), glutathione (GSH) and catalase (CAT) kits were purchased from Nanjing Jiancheng Biotechnology Co. Ltd (Nanjing, China).

Animals

Adult male Kunming mice (6 weeks old, weighing 18–23 g) were housed with free access to food and water. The animals were kept at regulated temperature (22 ± 2°C) and humidity (50 ± 10%) on a 12-hour light/dark cycle with ad libitum access to standard commercial food and pure water. After acclimating to laboratory conditions, the mice were divided at random into six groups of 10 animals each: control group (CT), stress group (ST), control + medium dose of luteolin group (CM), and three stress groups with low, medium and high doses of luteolin modulation, respectively (LD, MD and HD). The mice received humane care throughout the experiment according to the Chinese Council on Animal Care and Institutional Care Committee of Xuzhou Medical College. All efforts were made to minimize both the number of animals used and their suffering.

Drugs and treatment schedule

CT and ST groups of mice received 0.5% sodium carboxymethyl cellulose as vehicle treatment. Luteolin was suspended in 0.5% sodium carboxymethyl cellulose and daily administered intragastrically at doses of 25, 50 and 100 mg/kg 30 min before the animals were subjected to psychological stress for three consecutive weeks. The CM group received the medium dose of luteolin at the same time to that of LD, MD and HD groups, while was not received psychological stress treatments. All compounds were administered systemically in a volume of 0.01 mL/g body weight.

Psychological stress model

The method used in this study was a modified method for psychological stress as described previously, ^13,14 of which the animal model of psychological stress was developed by restraint for six hours daily in well-ventilated polypropylene tubes, with no physical suppression or pain experience in mice. The tubes were 10.5 cm in length long and 3.2 cm in diameter. Each tube was covered on one end with a Plexiglas covering, which had a small air hole (0.2 cm in diameter) in it. This protocol was scheduled for three consecutive weeks. During the fourth week, animals were subjected to step-through test, open-field test and subsequently followed by a Morris Water Maze (MWM) test. Thereafter, the animals were anesthetized with ether and killed by decapitation after aortic exsanguinations. Blood was collected and serum isolated from the whole blood. Brains were rapidly removed, and the prefrontal cortex and hippocampus tissues were isolated. The samples were stored at −80°C until processed for biochemical estimations.

Step-through test

One day after the end of the treatment with luteolin or vehicle, the step-through passive avoidance response was immediately performed according to previous study with minor modifications. ¹⁵ The step-through apparatus consists of six compartments of equal size (15 × 10 × 11 cm). Each compartment was divided into a light and a dark chamber by a guillotine door (3 cm in diameter). The light chamber was equipped with an illuminator. As described previously, ¹⁵ a single acquisition (training) trial was conducted prior to test. The trial consisted of a 30-s adaptation in one chamber which was dark at first, then followed by the onset of a bright light. To avoid the light, the mice crossed over into the darkened adjacent chamber and received a mild 0.8 mA footshock for one second. The time to crossover from the light chamber to the darkened chamber (latency) was recorded with a maximum trial length of 180 s. The mice were tested for recall 24 h after the acquisition period to determine whether they had been trained to criterion by remaining in the light chamber for 180 s to avoid the footshock. Any mice that did not meet the criterion were restrained. The latency and number of mistakes were used to measure the ability of the rats to remember adverse stimuli.

Open-field test

One day after the step-through test, the ambulatory behavior was assessed in an open-field test apparatus consisting of a square wooden arena (30 × 30 cm) marked with black lines. ¹⁶ The floor of the wooden arena was evenly divided into 6 × 6 cm squares marked with black lines. In the test, mice were individually placed in the center of the arena and permitted for free explorations. After 20-min adaptation, the number of squares crossed by the mice and the number of rearing on the hind paws were recorded during a test of three minutes. Between trials, the mouse was returned to its home cage in the same room, and open field was cleansed with a detergent and dried before occupancy by another mouse.

MWM test

Following the open-field test, the MWM test was carried out based on the method described previously ¹⁷ with minor modifications. The experimental apparatus consisted of a circular water tank (120 cm in diameter, 60 cm in height) located in a test room with several cues on white walls. With its inner surface painted black, the pool was filled with water to a height of 40 cm (maintained at 25 ± 1°C), covering an invisible (black) 10 cm square platform which was located approximately 1.5 cm below the surface of the water. The pool was virtually divided into four quadrants and the platform was placed at a fixed position in the center of a quadrant. Four different start points (NE, SE, SW and NW) were equally spaced around the circumference of the pool.

The MWM test lasted for five consecutive days following the 21 d of stress treatment, in which mice were trained in the MWM and their latency to platform was recorded with four trials per day over four days. At the beginning of each trial, the mice were released at one of the four possible starting points facing the wall, and allowed to swim freely until they reached the platform. If mouse did not find the escape platform within 90 s, it was given a latency score of 90 s. The inter-trail interval was 10 min. At the end of each session, all animals were dried and returned to their home cages. Twenty-four hours following the four consecutive days, the platform was removed. Mice were scored during a 90-s probe trial for latency to reach the platform and the time spent in the target quadrant to assess consolidation and retrieval of memory.

Brain homogenate

Following the behavior assessments, the animals were sacrificed with the brain removed, and the prefrontal cortex as well as hippocampus was isolated. To examine biochemical parameters, the prefrontal cortex and hippocampus were weighed, homogenized in nine volumes (1:9 w/v) of cold saline to prepare a 10% tissue homogenate in an ice bath and centrifuged at 1699 g for 15 min (Eppendorf Centrifuge 5804R, Hamburg, Germany). Thereafter, the supernatant was collected for the subsequent analysis of oxidative alterations.

Estimation of oxidative alterations in prefrontal cortex and hippocampus

The changes of MDA, SOD, GSH and CAT in prefrontal cortex and hippocampus were measured by their corresponding kits (Jiancheng Bio-tech, Nanjing, China) according to the manufacturer's instructions. Measurement in all assays described above was carried out in triplicates. The protein amount in all samples was measured by the bicinchoninic assay.

Blood samples collection

Blood samples were collected from the orbital sinus in 2 mL Eppendorf tubes and centrifuged at 1699 g for 10 min at 4°C to separate the serum from whole blood. The blood samples were stored at −80°C until assay.

Measurement of serum corticosterone, dopamine and norepinephrine concentrations

Corticosterone concentrations in the serum were measured using the Corticosterone ELISA kit (Adlitteram Diagnostic Laboratories) for mice according to the manufacturer's instructions. The kit uses a double-antibody sandwich ELISA to assay the level of mouse corticosterone in samples and the detection limit of the assay was 5 ng/mL. Similarly, the concentrations of dopamine and norepinephrine in serum samples were also respectively analyzed by their corresponding ELISA kits.

Statistical analysis

All statistical analyses were performed using the SPSS software, version 13.0 (SPSS Inc., Chicago, IL, USA). Values are expressed as mean ± SEM (standard error of the mean). For the MWM test, data in escape latency were analyzed by two-way analysis of variance (ANOVA), the factors being treatment and training day. Statistical significance was set at P < 0.05. For the open-field, step-through, biochemical and neuroendocrine assays, differences between groups were analyzed with one-way ANOVA followed by the Dunnett's post hoc test. Results were considered significant at P < 0.05.

Performances in step-through test of mice

In the acquisition trial, the step-through latencies did not differ among the five groups (P > 0.05). The step-through latency in the 24-h retention trail significantly decreased in the ST group in contrast to the CT group (P < 0.001). Treatment with luteolin (25, 50 and 100 mg/kg) significantly increased the step-through latency as compared with the ST group (P < 0.01 or P < 0.001, Figure 1). Treatment with luteolin did not affect the step-through latency during either the training or retension sessions for the mice without restraint stress treatments. OPEN IN VIEWER

Performances in open-field test of mice

Compared with the CT group, the crossing and rearing frequencies of the ST group in the open-field test were significant decreased, and these changes were improved in the LD group (P < 0.05). Furthermore, there were no remarkable difference in crossing and rearing frequencies of the MD and HD groups in contrast to that of the ST group (Figures 2a and b, P > 0.05). However, no significance in the crossings and rearings was found between mice treated with luteolin alone and the mice of the CT group. OPEN IN VIEWER

Performances in MWM test of mice

To examine the influences of luteolin on cognitive impairment induced by psychological stress, we tested the learning and memory using the MWM test and the results are shown in Figure 3a. The mean escape latency did not differ between any of the groups on the first day of testing in MWM test. While from the second day onwards, there was a significance difference in escape latency between CT and ST animals (P < 0.05). Chronic luteolin treatment significantly decreased the escape latency in stressed mice (Figure 3a). However, the mice in the HD group showed no significance in latency to reach the platform compared with the ST group (P > 0.05). OPEN IN VIEWER

To assess the consolidation of memory, the mice were subjected to a probe trial on day 5. During the probe trial, the time of mice staying in the platform quadrant was determined (Figure 3b). A significantly shorter stay was observed in the ST group, which was significantly reversed by LD and MD treatment (P < 0.05). In contrast, no significant difference was found in the time staying in the platform quadrant of HD treatment mice when compared with the ST group (P > 0.05).

Oxidative alterations in prefrontal cortex and hippocampus

Concentrations of serum corticosterone

Concentrations of serum norepinephrine and dopamine

Compared with the CT group, mice exposed to the psychological stress exhibited significant decline in both the serum norepinephrine and dopamine concentrations (P < 0.01 or P < 0.001). Additionally, the concentration of norepinephrine was increased in the LD group in contrast to that of the SD group (P < 0.05). Furthermore, the serum dopamine concentrations were observed to be significantly higher in LD and HD groups when compared with the CT group (P < 0.05). However, luteolin did not alter the levels of norepinephrine and dopamine in the serum of mice without restraint stress treatments (P > 0.05, Table 3).