Baicalein prevents stress-induced anxiety behaviors in zebrafish model

Abstract

Baicalein is a flavonoid mainly obtained from plants with wide range of biological activities, including neuroprotection. An acute and unexpected chronic stress (UCS) protocol has recently been adapted to zebrafish, a popular vertebrate model in brain research. The present study was aimed to evaluate baicalein's anti-anxiety potential in a zebrafish model by induction, which included neuropharmacological evaluation to determine behavioural parameters in the novel tank diving test (NTDT) and light-dark preference test (LDPT). The toxicity was also assessed using the brine shrimp lethality assay, and the 50% lethal concentration (LC₅₀) was determined. The animals were then stressed for 7 days before being treated with different doses of baicalein (1 and 2 mg/L) for another 7 days in UCS condition. Due to acute stress and UCS, the frequency of entries and time spent in the 1) top region and 2) light area of the novel tank reduced significantly, indicating the existence of elevated anxiety levels. The biological activity of baicalein was demonstrated by its high LC₅₀ values (1,000 μg/ml). Additionally, baicalein administration increased the frequency of entries and duration spent in the light region, indicating a significant decrease in anxiety levels. Overall, the present results showed that baicalein has a therapeutic advantage in reversing the detrimental consequences of UCS and acute stress, making it is a promising lead molecule for new drug design, development, and therapy for stress.

Keywords: anti-anxiety; anti-stress; baicalein; neuroprotection; zebrafish.

FIGURE 1

Baicalein, a bioactive molecule found in Scutellaria baicalensis Georgi and Oroxylum indicum (L.) Kurz, has been shown to be effective in the treatment of neuropsychiatric diseases, particularly anxiety occuring as a result of 1) dysregulation of hormones released from the hypothalamic-pituitary-adrenal (HPA) axis, which when aggravated by a stressor result in chronic oxidative stress and neurotoxicity. It has been suggested that the selective serotonin reuptake inhibitor (SSRI) fluoxetine, which helps to upregulate GABA receptors, may work through a similar mechanism to baicalein to prevent seizures. 2) Baicalein reverses the decline in the expression of the receptors hence may prevent neuronal atrophy. Abbreviations: ACTH, Adrenocorticotropin; SOD, Superoxide dismutase; CAT, Catalase; ROS, Reactive oxygen species; GAT1, GABA transporter 1.

FIGURE 2

Study timeline of unpredictable chronic stress: Group I- control (non-stressed), Group II -UCS (stressed), Group III- UCS + Diazepam (US1), Group IV- UCS + Group V- Fluoxetine (US2), Group VI- UCS + Group VII- Baicalein conc1 (UB1), Group VIII- UCS + Baicalein conc2 (UB2).

FIGURE 3

Brine shrimp lethality at 24 h in a 24-well plate. Plate with (A) sample and (B) brine shrimp.

FIGURE 4

Brine shrimp lethality assay for baicalein.

FIGURE 5

(A) Novel tank test of drug-treated group showing the exploratory behaviour of zebrafish (UCS) (B) The effect of treatment on time spent in the upper level of the tank in novel tank (UCS) (C) The effect of treatment on crossing of zones in novel tank (UCS) (D) The effect of treatment on total distance travelled in novel tank (UCS).Values were expressed as mean ± S.E.M. A one-way ANOVA followed by Bonferroni post hoc test (n = 8–10) was used. (A) Comparisons were made between the stressed group (UCS) with normal control. (B) Comparisons were made between US1, US2, UB1, and UB2 with stressed group (UCS). p-value ***represents p < 0.001, **represents p < 0.01, *represents p < 0.05, ^## represents p < 0.01. (B) shows the influence of baicalein, diazepam and fluoxetine on behavioral parameters in zebrafish subjected to UCS. As expected, UCS increased the time spent in the bottom area and decreased the entries as well as the time in the top area. The control group (control) showed the time spent in the upper zone (55 s) and the stressed group (UCS) showed decreased in the time spent in the upper zone (42 s). Baicalein-treated groups (UB1 andUB2) were found to explore more in the upper levels of the tank following transfer to a novel tank in comparison to standard drugs. On the other hand, UB1 group has increased time spent (47 s) while the UB2 group had 54 s as the time spent [panel (B)]. Fluoxetine and diazepam-treated group exhibited increased in the time spent in the upper levels (at 48 s and 52 s respectively). The total distance travelled [panel (C)] was not significantly affected by the unpredictable chronic stress model but the number of crossing [panel (D)] was decreased by the UCS protocol.

FIGURE 6

(A) Novel tank test of drug-treated group showing the exploratory behaviour of zebrafish (AS) (B) The effect of treatment on time spent in the upper level of the tank in novel tank (AS) (C) The effect of treatment on crossing of zones in novel tank (AS) (D) The effect of treatment on total distance travelled in novel tank (AS). Values are presented as the mean ± S.E.M. A one-way ANOVA followed by Bonferroni post hoc test (n = 8–10) was used. (A) Comparisons were made between the stressed group (AS) with the normal control. (B) Comparisons were made between AS1, AS2, AB1 andAB2 with the stressed group (AS). p-value ***represents p < 0.001, **represents p < 0.01, *represents p < 0.05, ^## represents p < 0.01. (B) shows the effects of Baicalein (AB1 andAB2), diazepam (AS1) and fluoxetine (AS2) in zebrafishes involved in the acute stress model. As expected, diazepam significantly decreased the time spent in the bottom and increased the time spent the upper zone of the tank [panel (B)]. Baicalein-treated groups such as AB2 and AB2 significantly increase the time spent in the upper zone (38 and 42 s respectively). The distance travelled [panel (D)], the number of crossings [panel (C)] and entries to the bottom area was not affected by any intervention.

FIGURE 7

(A) Light dark test of drug-treated group of zebrafish (UCS) (B) The effect of treatment on the time spent in the light zone of the tank (UCS) (C) The effect of treatment on entries to the light zone of the tank (UCS). The data are presented as the mean ± S.E.M. A one-way ANOVA followed by a Bonferroni post hoc test was used (n = 8–10). (A) Comparisons were made between the stressed group (UCS) with the normal control. (B) Comparisons were made between US1, US2, UB1 and UB2 with the stressed group (UCS). p-value ***represents p < 0.001, **represents p < 0.01, *represents p < 0.05, ^## represents p < 0.01.

FIGURE 8

(A) Light dark test of drug-treated group of zebrafish (AS) (B) The effect of treatment on time spent in the light zone of the tank (AS) (C) The effect of treatment on entries to the light zone of the tank (AS). Values are presented as the mean ± S.E.M. A one-way ANOVA followed by Bonferroni post hoc test was used (n = 8–10). (A) Comparisons were made between the Stressed group (AS) with normal control. (B) Comparisons were made between AS1, AS2, and AB1 andAB2 with the stressed group (AS). p-value ***represents p < 0.001, **represents p < 0.01, *represents p < 0.05, ^## represents p < 0.01, ns represents being not significant.

FIGURE 9

Binding interactions of (A) baicalein with GABA(A)receptor (PDB ID: 6X3X) and (B) baicalein with serotonin transporter (PDB ID: 5I73).

FIGURE 10

The acute stress protocol induced locomotor changes that were reversed by diazepam (DZP) and fluoxetine treatments (FLU). The hypothalamic-pituitary-adrenal (HPA) axis results in a rise of corticosteroids levels in the blood, which are subsequently delivered to the spleen and periphery, where they decrease a number of immune processes. FLU and DZP both can to act as anxiolytics by decreasing cortisol responses to acute stress. Abbreviations: ACTH, Adrenocorticotropin; PRL, Prolactin; hGH, human growth hormone; IL-1, Interleukin-1.