Genistein acts as antidepressant agent against chronic mild stress‐induced depression model of rats through augmentation of brain‐derived neurotrophic factor

Abstract In this study, the antidepression effects of genistein were investigated in rats induced with chronic mild stress. Animals were designated into the following groups: normal control, control, 10 mg, and 100 mg. The dose was given for 45 consecutive days via the oral route. Sucrose preference analysis, forced swim, and open field tests were performed, and serum cortisol and monoamine levels in brain tissue were determined. The mRNA and protein expression of brain‐derived neurotrophic factor (BDNF) was also examined. Supplementation with genistein significantly increased the sucrose preference ratio, locomotor activity, and monoamines and decreased serum cortisol levels. The mRNA expression of BDNF in the brain tissue was substantially reduced by 0.73% in control rats. However, supplementation with genistein significantly increased BDNF mRNA expression (by 107% and 229.6% in groups 10 mg and 100 mg, respectively). Similarly, the protein expression of BDNF increased by 82.3% and 141.2% in groups 10 mg and 100 mg, respectively. Taken together, these results suggest that supplementation with genistein may be effective against depression.


INTRODUCTION
Depression is a state of aversion to activity and low mood, and affects behavior, thoughts, feelings, sense of well-being, and tendencies (Planchez et al., 2019). Depression is a chronic, recurring, and severe life-threatening illness that affects people globally (Du et al., 2020). It can also be a side effect of medical treatments and physical exercise or a symptom of dysthymia (Rahmati et al., 2017). Freitas et al. (2013) have reported that the monoamine oxidase A, tricyclic antidepressants, and specific serotonin and noradrenaline reuptake inhibitors are medically available drugs for the treatment of depression. However, weight gain, sleep disorder, sexual dysfunction, cardiac toxicity and hypokine-This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. © 2021 The Authors. Brain and Behavior published by Wiley Periodicals LLC sia are main side effects of these drugs (Jiang et al., 2017). Therefore, potential agents are required for the treatment of depression without any main side effects.
Genistein is trihydroxyisoflavone found in soybeans and known to have estrogenic activity (Shen et al., 2018). Genistein has been extensively investigated due to its effects on osteoporosis, cancer, cardiovascular diseases, and depression (Atteritano et al., 2014;Kageyama et al., 2010;Marini et, al.,2010). Researchers have recommended the genistein as remedy for the menopausal symptoms and osteoporosis (Dixon & Ferreira, 2002). Researchers have reported that antidepressant-activity through reduction of immobility time in forced swimming test (Sapronov & Kasakova, 2008), and similar Brain Behav. 2021;11:e2300. wileyonlinelibrary.com/journal/brb3 1 of 7 https://doi.org/10.1002/brb3.2300 effect was observed in ovariectomized female rats, suggesting the strong antidepressant-activity of genistein. However, the mechanism of action of genistein against depression is yet to be investigated. Thus, the present study evaluated the effect of genistein on chronic mild stress-induced depression in male albino rats.

Rats
Male

Induction of chronic mild stress
Chronic mild stress was induced in rats according to a previously described procedure with slight modifications (Willner, 2016). Briefly, animals were trained to consume 1% sucrose solution before apply- The procedure was repeated for 28 days.

Groups and treatments
Rats were designated into four groups: normal control, control, 10 mg/kg of genistein, and 100 mg/kg of genistein. The dose was given for 45 consecutive days via the oral route. Each group contained six rats.

Sucrose preference analysis
The sucrose preference test was performed as previously described (He et al., 2020). Briefly, the two bottles of sucrose solution (1%) were placed on the rat cage separately. Then, free access was provided to the rats to drink water from these cages for 1 day. Then, one bottle was continued with the same sucrose solution and another bottle was filled with water for the next 24 h. Bottle position was changed to avoid the influence of bottle position. Then, all the animals were deprived of water for another 23 h, and sucrose preference examination was performed for each rat. Then, the amount of water and sucrose solution consumed was recorded and calculated.

Behavioral test
Forced swim and open field tests were performed as previously described (Valencia et al., 2019). All the rats were placed in the cen-

Determination of serum cortisol
The serum cortisol level was estimated using an enzyme-linked immunosorbent assay (ab108665, Abcam, Cambridge, UK). At the end of the treatment, rats were dissected and blood was collected and processed with the serum for the determination of cortisol level.
The clear supernatant was collected, and monoamine levels were determined by using a radioimmunoassay kit (Abcam).

RT-PCR
The total RNA was extracted from the homogenates of brain tissues. Then, RNA pellets were washed in diethyl pyrocarbonate-treated water; gel electrophoresis was carried out to check RNA integrity. The RNA purity was calculated by measuring at 260 nm. Total RNA was transcribed into cDNA by adding 0.5 ng of oligo dT primer and 1 µg of total RNA in a thermal cycler for 10 min at 65 • C. Finally, 5× RT buffer (4 µl), 10 mM dNTPs (2 µl), and reverse transcriptase (100 U) were added to PCR tubes and incubated in a thermal cycler for 60 min at 37 • C and then for 10 min at 90 • C. Brain-derived neurotrophic factor (BDNF) mRNA expression was determined by RT-PCR (Table 1). The relative ratios of BDNF mRNA expression were determined according to the 2 -∆∆CT method (Mokhtari et al., 2017).

Immunofluorescence
The brain was dissected and sectioned. Then, sections were fixed in formalin and embedded in paraffin. Then, the sections were deparaffinized, rehydrated with xylene with graded alcohol series. The 3% hydrogen peroxide was used to inhibit endogenous peroxidase activity.
Bovine serum albumin (2%) was used to block nonspecific binding sites. Brain tissue was treated with an anti-BDNF (1:300 dilutions; ab226843; Abcam) antibody overnight and then incubated with TA B L E 1 List of primers used in RT-PCR reaction for the amplification of brain-derived neurotrophic factor

Statistical analysis
Data are presented as the mean ± standard deviation. The means were compared using one-way analysis of variance (ANOVA), followed by Tukey's post hoc test. Differences were considered significant at p < .05.

RESULTS
The present study evaluated the protective effect of genistein on chronic mild stress-induced depression. Figure 1 shows the sucrose preference ratio of control and genistein-treated rats. The sucrose preference ratio was substantially reduced by 110.9% in control rats compared to normal control rats. However, supplementation with genistein significantly increased the sucrose preference ratio to 21.7% and 102.2% at 10 and 100 mg/kg of genistein, respectively (Figure 1, p < .05).
Behavioral parameters, such as rearing, crossing, and immobility time, were determined in control and genistein-treated rats. Rearing  (e.g., 5-HT, noradrenaline, and 5-HIAA) was substantially reduced in brain tissue homogenate. However, supplementation with genistein significantly increased these monoamine levels to near-normal levels (Table 2, p< .05). In brain tissue, BDNF mRNA was substantially reduced by 68% in the control rats. However, supplementation with genistein significantly increased BDNF mRNA expression by 40.6% and 187.5% at 10 and 100 mg/kg of genistein, respectively (Figure 4, p< .05). Similarly, the protein expression of BDNF was increased by 82.3% and 141.2% at 10 and 100 mg/kg of genistein, respectively ( Figure 5, p< .05).

DISCUSSION
The present study evaluated the protective effect of genistein on chronic mild stress-induced depression. Plants and plant-derived agents have recently attracted the attention of researchers for their therapeutic effects against several illnesses, including mental disorders (Ekor, 2014). Researchers have reported that various herbal plants and herbal formulations are useful against experimental depression (Jawaid et al., 2011). Genistein has been extensively investigated due to its effects on osteoporosis, cancer, cardiovascular diseases, and F I G U R E 3 Genistein decreases the serum cortisol in an experimental model of chronic mild stress-induced depression. **p< .01 versus normal control and ## p < .01 versus control F I G U R E 4 Genistein increases the brain-derived neurotrophic factor (BDNF) mRNA expression in an experimental model of chronic mild stress-induced depression. ***p< .001 versus normal control, # p< .05 and ## p < .01 versus control depression (Atteritano et al., 2014;Kageyama et al., 2010;Marini et al., 2010). Researchers have recommended the genistein as remedy for the menopausal symptoms and osteoporosis (Dixon & Ferreira, 2002).
Researchers have reported that antidepressant-activity through reduction of immobility time in forced swimming test (Sapronov & Kasakova, 2008), and similar effect was observed in ovariectomized female rats, suggesting the strong antidepressant-activity of genistein. Researchers have reported that an increased level of serum cortisol leads to severe behavioral alterations, such as depression (Busquet et al., 2010 (Hurley et al., 2013). Taken together, these results suggest that supplementation with genistein can be useful against depression.

COMPETING INTERESTS
All authors declare that they have no conflict of interests.

CONSENT FOR PUBLICATION
This article does not contain any studies involving human participants performed by any of the authors.

AUTHORS' CONTRIBUTIONS
MC conceived and designed the research. LZ and HD performed experiments and statistical analysis. LS wrote the manuscript. All authors read and approved the final manuscript.

AVAILABILITY OF DATA AND MATERIALS
The data and material during the current study are available from the corresponding author on reasonable request.