Breviscapine Ameliorates Fear Extinction and Anxiety in BALB/cJ Mice
Article information
Abstract
Objective
In this research, the influence of breviscapine on anxiety, fear elimination, and aggression and the potential mechanism was investigated.
Methods
Anxiety and locomotion were analyzed by elevated plus maze and open field test in mice. Bussey-Saksida Mouse Touch Screen Chambers were used to perform fear conditioning. Territorial aggression was assessed by resident intruder test. Protein levels were evaluated by Western blot. Breviscapine improved fear-extinction learning in BALB/cJ mice.
Results
Breviscapine at 20–100 mg/kg increased center cross number, total distance traveled, and velocity in a dose-dependent manner. On the other hand, breviscapine at 20–100 mg/kg decreased the immobility time in open field test. In addition, breviscapine at 20–100 mg/kg increased the ratio of time on the open arm, time on the distal parts of the open arm, and total distance traveled in elevated plus maze. Breviscapine at 100 mg/kg increased the average attack latency and decreased the number of attacks over the last 3 days of resident intruder test. In hippocampus, protein levels of postsynaptic density protein-95 and synaptophysin were elevated by breviscapine at these three doses.
Conclusion
The administration of breviscapine alleviates fear extinction, anxiety, and aggression, while increases locomotor in a dose-dependent manner, which might be associated with its influence on synaptic function.
INTRODUCTION
Breviscapine is extracted from the dried whole herb of Erigeron breviscapus (Vant) hand-mazz [1]. Breviscapine is complex of several flavonoids, which is mainly composed of breviscapine (4'-hydroxy baicalein-7-o-glucuronide) and a small amount of breviscapine (apigenin-7-o-glucuronide) [2]. Studies have shown that breviscapine has the ability to protect neurons and prevent cytotoxicity, regulate vascular endothelial function, relieve cerebrovascular spasm, improve microcirculation, reduce blood lipids, regulate immunity and reduce inflammatory response, anti-free radical damage, and inhibit platelet agglutination [2]. Breviscapine has good efficacy in cardiovascular and cerebrovascular disease therapy, however, the mechanism by which breviscapine exerts its therapeutic effect is still unclear.
Adolescents with attention deficit hyperactivity disorder (ADHD) and conduct disorder (CD) exhibit aggressive and antisocial behavior and have become a growing social and socioeconomic problem. In addition to heightened aggression, these adolescent patients often have increased anxiety, hyperactivity, attention deficit, impulsivity, and impaired fear memory extinction [3]. To date, finding the right pharmacological treatment remains a challenge.
BALB/cJ mouse models have similar characteristics to CD and ADHD, including increased aggression and inattention [4]. Due to its neuroprotective effects, breviscapine may also be useful in CD and ADHD treatment. This study investigated the effects of breviscapine on BALB/cJ mice behavior, mainly including aggression, anxiety and fear processing/elimination.
METHODS
Animals
Male C57BL/6J mice (6 weeks) were housed in groups of six mice and male BALB/cJ mice (8 weeks) were housed individually under a reversed light/dark cycle (12/12 h). Experimental procedures were approved by the Committee of Animal Experiments of the Fourth Military Medical University (IRB No. 20210305DL27).
Breviscapine (Sigma, St. Louis, MO, USA) was dissolved in 4% dimethylsulfoxide (DMSO) and administrated through intraperitoneally injection. BALB/cJ mice were separated into four groups. Mice in vehicle group were treated with DMSO daily. Mice in were administrated with 20, 50, or 100 mg/kg breviscapine daily. Schedule of the study design was shown in Figure 1A. All the tests were performed in the first hour of darkness and drug administration was performed 20 min before the test.
Breviscapine (Bre) alleviates anxiety in the open field test (N=12). (A) Schedule of the study design. Timeline of the different experiments. All the tests were performed in the first hour of darkness and drug administration was performed 20 min before the test. Breviscapine increased center cross number (B), total distance (C), and velocity (D). On the other hand, breviscapine decreased the immobility time (E). Data were presented as mean±standard deviation. *p<0.05; **p<0.01; ***p<0.001 compared to vehicle. One-way analysis of variance followed Dunn’s multiple comparisons test.
Open field test
An activity chamber (55×55×36 cm) was used for the quantifying the locomotion activity. Locomotion activity during 5 min was recorded after being placed in center. The field was divided into four quadrants. The number of center cross, total distance traveled, velocity, and immobility time were recorded.
Elevated plus maze
A central platform (6×6 cm) was placed 60 cm above the ground. Two open arms (36×6 cm) and two closed arms (36×15×6 cm) were linked with the central platform. The test was performed under dim-light condition in the dark phase and the behavior was recorded within 5 min. The time spent on closed arms, open arms, and center platform and the total distance traveled were analyzed.
Fear conditioning
Bussey-Saksida chambers were used to perform fear conditioning. Manual shocks were provided through grid floor in the chambers. In fear conditioning, animals received 30 s auditory cue for three times, 0.7 mA foot shock was provided in the last 2 seconds. In extinction learning, animals received 30 s auditory cue for 15 times without the shock. In extinction test, animals received 30 s auditory cue for 15 times without the shock. The freezing time and beam breaks numbers during extinction learning and extinction test were analyzed.
Resident intruder test
Territorial aggression was assessed in 5 days by resident intruder test. The test was performed in BALB/cJ mice cages. An unfamiliar C57BL/6J intruder mouse was randomly assigned to BALB/cJ mouse on each testing day. During the first 5 min, two animals were separated by a transparent Plexiglas screen but with auditory, visual, and olfactory perception. Then the screen was removed, and animal activity was recorded in 5 min. C57BL/6J mouse was removed after the test. The average attack latency and number of attacks were analyzed.
Western blot
In this research, Western blot was performed following standard protocol. Following primary antibodies were used: anti-synaptophysin (SYP) (Abcam, Cambridge, MA, USA), anti-postsynaptic density protein-95 (PSD-95) (Abcam), and GAPDH (Abcam). GAPDH was used as a loading control.
Statistical analysis
Data were analyzed by SPSS software 20.0 (IBM Corp., Armonk, NY, USA) and presented as mean±standard deviation. Data were analyzed by one-way analysis of variance (ANOVA) followed Dunn’s multiple comparisons test, or two-way ANOVA followed Tukey’s multiple comparisons test. It was regarded as statistically significant when p-value was less than 0.05.
RESULTS
Breviscapine alleviates anxiety-related behavior and increases locomotor in BALB/cJ mice
Anxiety and locomotion were assessed by the open-field test. Compared with those in the Vehicle group, mice treated by breviscapine at the doses of 20–100 mg/kg had increased center cross number, the total distance traveled, and velocity (Figure 1B-E) in a dose-dependent manner. On the other hand, breviscapine significantly declined the immobility time in the open-field test (Figure 2D). The effects were the most obvious when the dose was 100 mg/kg.
Breviscapine (Bre) alleviates anxiety in the elevated plus maze (N=12). Breviscapine increased the time spent on the open arms as a ratio of the time spent on both the closed and open arms (A), time spent in the distal parts of the open arms (C) as well as total distance traveled (D). Breviscapine decreased the time spent in center (B). Data were presented as mean±standard deviation. *p<0.05; **p<0.01; ***p<0.001 compared to vehicle. One-way analysis of variance followed Dunn’s multiple comparisons test.
After being treated with either dosage of breviscapine, BALB/cJ mice had higher ratio between the duration spent in the open and closed arms and longer time spent in the distal parts of the open arms compared to mice treated with vehicle (Figure 2A and C). BALB/cJ mice treated with 50 mg/kg or 100 mg/kg breviscapine spent less time on central platform than mice in the Vehicle group (Figure 2B). Additionally, the administration of 50 mg/kg or 100 mg/kg breviscapine significantly elevated the total distance traveled in maze of BALB/cJ mice (Figure 2D). These results suggest that breviscapine alleviates anxiety-related behavior and increases locomotor in a dose-dependent manner.
Breviscapine improves fear-extinction in BALB/cJ mice
The influence of breviscapine on mixed-cue fear conditioning in BALB/cJ mice was assessed in this research. During both fear extinction learning and extinction test, administration of breviscapine decreased the freezing time in BALB/cJ mice in a dose dependent manner (Figure 3A and B). Meanwhile, BALB/cJ mice treated with 50 or 100 mg/kg breviscapine had significant elevation in the number of beam breaks when compared with those in the Vehicle group during both fear extinction learning and extinction test (Figure 3C and D).
Breviscapine (Bre) improves fear-extinction learning (N=12). The time spent freezing in extinction learning (A) and extinction test (B). Cumulative beam breaks number in extinction learning (C) and extinction test (D). Data were presented as mean±standard deviation. *p<0.05; **p<0.01; ***p<0.001 compared to vehicle. One-way analysis of variance followed Dunn’s multiple comparisons test and two-way analysis of variance followed Tukey’s multiple comparisons test.
Breviscapine alleviates aggression behavior in BALB/cJ mice
The aggression in BALB/cJ mice was assessed by the resident intruder test. As shown in Figure 4A, the administration of 50 or 100 mg/kg breviscapine significantly increased the average attack latency over the last 3 days of test. The number of attacks over the last 3 days of test was significantly decreased in the 100 mg/kg breviscapine group when compared with the Vehicle group (Figure 4B).
Breviscapine (Bre) alleviates aggression behavior (N=12). The average attack latency (A) and number of attack (B) over the last 3 days of tests. Data were presented as mean±standard deviation. *p<0.05 compared to vehicle. One-way analysis of variance followed Dunn’s multiple comparisons test.
Breviscapine increases the synaptic proteins levels in the hippocampus of BALB/cJ mice
In this research, we also analyzed the influence of breviscapine on the synaptic proteins in the hippocampus of BALB/cJ mice. When compared with mice in the Vehicle group, those treated by breviscapine at 20–100 mg/kg doses had increased protein levels of both PSD-95 and SYP (Figure 5), and 100 mg/kg breviscapine group had the highest levels of PSD-95 and SYP expression.
Breviscapine (Bre) increased the levels of PSD-95 and SYP in hippocampus of BALB/cJ mice (N=3 from 12 mixed tissue homogenate). (A) Western blot result of PSD-95 and SYP in hippocampus from different groups the relative expression of PSD-95 (B) and SYP (C). Data were presented as mean±standard deviation. *p<0.05; **p<0.01; ***p<0.001 compared to vehicle. One-way analysis of variance followed Dunn’s multiple comparisons test.
DISCUSSION
ADHD is a common chronic neurobehavioral disorder [5]. ADHD has a prevalence of 3%–5% in school-aged children in US and 5.95%–11.5% in China [6,7] ADHD is a serious disorder that affects academic and vocational achievement, family and social functioning of children and is often associated CD. 70%–85% of children with ADHD have symptoms that persist into adolescence and, in some cases, into adulthood, with a tendency to develop antisocial personality, CD, substance use disorder, and substance abuse [8,9]. ADHD has become an important public health problem and has attracted widespread attention from the whole society. Currently, methylphenidate is preferred for ADHD therapy, but it has adverse effects such as loss of appetite, induced twitching, drug dependence, and increased heart rate, and the symptoms tend to rebound after discontinuing the drug, which brings some limitations to clinical use [10].
Flavonoids have been shown to have antioxidant effects [11]. Breviscapine is proved to perform neuroprotective effects in APP/PS1 mice [12]. In transient cerebral ischemia/reperfusion rat model, cognitive impairment is alleviated by breviscapine through its antioxidant and anti-inflammatory effects [13]. It is reported that breviscapine ameliorates the learning and memory deficits of Alzheimer’s disease mice [14]. Thus, breviscapine may also have positive effects in the therapy of ADHD and CD thought its neuroprotective effects.
It has been reported that BALB/cJ mouse model has similar phenotypes in ADHD and CD, such as inhibited social behavior, enhanced inattention, and elevated aggression [4]. In this research, we explored the effects of breviscapine on phenotypes in ADHD and CD in BALB/cJ mice.
Children with ADHD exhibit significant emotional problems with a probability of concomitant anxiety and depression [15]. Anxiety in patients with ADHD may be associated with a decrease in daily living skills due to cognitive deficit [16]. There is a high co-morbidity between ADHD and CD, which leads to stronger negative emotions in patients.
In BALB/cJ mice, results demonstrated that breviscapine administration had an anxiolytic action. Breviscapine increased center cross number, the total distance traveled, and the velocity but decreased the immobility time in open field test. Breviscapine increased the total distance traveled and the time in the open arms but decreased the time in center of the maze.
Non-traumatized, unmedicated adult ADHD patients showed abnormalities in fear circuits. Subjects with ADHD had greater activation in the insular cortex during early extinction, less activation in the ventromedial prefrontal cortex and the dorsal anterior cingulate cortex during late extinction learning [17]. In rodents, when the animals are afraid, one of the natural defense responses is freezing. In fear conditioning training, the pairing of sound and light signals (neutral conditioned stimuli) and electric shock feet (aversive unconditioned stimuli) elicits a fear response in the animal [18]. In this research, BALB/cJ mice administrated with 50 or 100 mg/kg breviscapine had significantly decreased freezing time and increased beam break number. Thus, the administration of breviscapine enhanced fear-extinction.
ADHD is often correlated with aggression. Aggression may be a distinct symptom or generated from impulsivity and hyperactivity [19]. C57BL/6J mice are in general less anxious than BALB/cJ mice [20]. Resident intruder test was performed to evaluate the influence of breviscapine on aggression. In this research, only the treatment of 100 mg/kg breviscapine significantly increased the average attack latency and decreased the number of attacks over the last 3 days of tests.
In APP/PS1 transgenic mice, breviscapine ameliorates Alzheimer’s disease-like symptom by reducing Aβ and proinflammatory cytokine levels and the inhibition of neuroinflammation [21]. We also explored the possible molecular mechanisms of breviscapine in alleviating ADHD and CD symptoms in BALB/cJ mice. Neurotransmitter dysfunction has been proved to participate in the development of ADHD. SYP regulates the release of neurotransmitter and the plasticity of synapse. The pathogenesis of ADHD is reported to have association with SYP [22]. PSD-95 is scaffolding protein which mainly expressed in postsynaptic densities [23]. N-methyl-D-aspartate receptor (NMDAR) anchored to the synaptic membrane by the interaction with PSD-95 [24]. ADHD-like behaviors can be induced by the abnormal function of NMDAR/PSD-95 complex [25]. In this research, we also analyzed the influence of breviscapine on the protein levels of SYP and PSD-95 in hippocampus of BALB/cJ mice. The protein levels PSD-95 and SYP in hippocampus of BALB/cJ mice were significantly elevated by the administration of breviscapine. Thus, breviscapine may alleviate ADHD and CD symptoms in BALB/cJ mice through its influence on synapse function.
In conclusion, the administration of breviscapine in BALB/cJ mice alleviated fear extinction, anxiety, and aggression, while increases locomotor dependent on its dose. The effects of breviscapine in BALB/cJ mice might be associated with its influence on synapse function. Thus, the treatment of breviscapine could be used as a potential strategy for the therapy of ADHD and CD.
Notes
Availability of Data and Material
The data could be obtained upon reasonable request to the corresponding author.
Conflicts of Interest
The authors have no potential conflicts of interest to disclose.
Author Contributions
Conceptualization: Wei Liang, Qun Yang, Fengzhan Li. Data curation: Wei Liang, Jing Huang, Shuya Yang, Chen Chen, Qun Yang, Fengzhan Li. Formal analysis: Wei Liang, Fengzhan Li. Funding acquisition: Fengzhan Li. Investigation: Wei Liang. Methodology: Fengzhan Li. Project administration: Fengzhan Li. Resources: Wei Liang. Software: Jing Huang, Shuya Yang, Chen Chen. Supervision: Fengzhan Li. Validation: Wei Liang, Qun Yang, Fengzhan Li. Visualization: Shuya Yang, Peng Huang, Jun Chen. Writing—original draft: Wei Liang. Writing—review & editing: Qun Yang, Fengzhan Li.
Funding Statement
None