A red pomegranate fruit extract-based formula ameliorates anxiety/depression-like behaviors via enhancing serotonin (5-HT) synthesis in C57BL/6 male mice

Xiping Zhu, Dongxio Sun-Wterhouse, Chun Cui,c,*

a College of Food Science and Engineering, South China University of Technology, Guangzhou 510640, Guangdong, China

b College of Biological and Food Engineering, Anhui Polytechnic University, Wuhu 241000, Anhui, China

c Guangdong Weiwei Biotechnology Co., LTD., Guangzhou 510640, China

Keywords:

Behavioral tests

Red pomegranate fruit extract

5-HT

Anti-inflammatory

Anxiety/depression

ABSTRACT

This study investigated the anti-anxiety/anti-depression potential of a formula containing red pomegranate fruit extract (RPFE; 40%, m/m), Lactobacillus rhamnosus (JB-1) (34%, m/m), magnesium gluconate (25%, m/m)and vanillin (1%, m/m). The RPFE formula (dose: 2.0, 1.5 or 1.0 mg/g·day) reversed behavioral dysfunctions and body weight gain induced by chronic restraint stress combined with corticosterone injection in C57BL/6 male mice. The RPFE formula exhibited the abilities to normalize the levels of serum inflammatory cytokines(NF-κB, TNF-α, IL-6, IL-1β and IFN-γ) and malondialdehyde (MDA), and activities of superoxide dismutase(SOD), catalase (CAT) and nitric oxide synthase (NOS), as well as relieve the injury of hippocampal neurons. The serotonin (5-HT) levels in hippocampus were increasingly enhanced, which might be mediated by reducing the activity of indoleamine-2,3-dioxygenase (IDO) and increasing the activity of tryptophan hydroxylase (TPH). Thus, the neuroprotective and ameliorating effects on anxiety/depression-like behaviors resulting from the RPFE formula ingestion were possibly related to serotonergic activation, which might be mediated via anti-inflammatory and anti-oxidant actions.

1. Introduction

World Health Organization (WHO) describes anxiety and depression as two highly prevalent and comorbid mental disorders,with anxiety disorders as “a group of mental disorders characterized by feelings of anxiety and fear”, and depressive disorders as “an illness characterized by sadness, loss of interest or pleasure, feelings of guilt or low self-worth, disturbed sleep or appetite, feelings of tiredness, and poor concentration”. Anxiety and depression affects humans differently and may coexist in the same individual concurrently or at different times. The co-occurrence of anxiety and depression likely causes a reduced rate of recovery and increased rate of recurrence. Although various medications are available in the treatment of these disorders including antidepressant and antianxiety drugs like benzodiazepines (for short-term use) and selective serotonin reuptake inhibitors (SSRIs) (for long-term use), almost a half of the patients with anxiety/depression are unwilling to take these drugs due to their side effects and toxicity in overdose [1]. Therefore,alternative therapeutics such as plant extracts and herbal products with high anti-depressant activities but low cost and minimal side effects, are increasingly sought.

Pomegranates with high concentrations of polyphenols are centuries-old fruit that have been utilized since ancient times and recently recognized as super fruits in early 2000s for their nutritional and medicinal properties. A number of studies reported that pomegranate extract supplementation with diet shows antiinflammatory properties [2] and significantly reduced oxidative stress in brain [3]. Furthermore, Ahn et al. [4] indicated that pomegranate extract (40%,m/m) improved depression and anxiety in a postmenopausal model with ovariectomized rats. Using 200 μL(1.67 × 109CFU) ofLactobacillus rhamnosus(JB-1) with increased immunity and nutrient absorption for animal gavage can significantly decrease stress-induced anxiety-like behavior [5]. Magnesium (a wide role in brain biochemistry) deficiency could cause depression and case histories are presented showing rapid recovery from major depression using 125-300 mg of magnesium supplementation [6]. Vanillin(4-hydroxy-3-methoxybenzaldehyde) has widely been accepted as a pleasant aroma, and was found to capable of evoking positive mood[7] alleviating depressive-like behaviorsviathe olfactory pathway in a rat model of chronic depression [8]. Vanillin is known to possess important anti-oxidation/anti-inflammatory activity and elevate serotonin levels in brain tissue [8,9]. However, anxiety/depression as a highly complicated psychiatric illness, the etiopathology as well as the clear mechanisms underlying its disorders are still far from understood. The above 4 single components have anti-anxiety or antidepression effects through different mechanisms, but no research has been reported on the combination of these 4 components for anti-anxiety or anti-depression. Accordingly, it is of high interest to examine whether or not formulas containing a red pomegranate extract, JB-1, magnesium and vanillin could act on anti-anxiety/depressant effect.

Excessive oxidative and nitrosative stress and inflammation[10] and metabolic disorders [11] are closely associated with the pathogenesis of anxiety/depression. Oxidative and nitrosative stress(O&NS) and immune-inflammatory response is influencing factors for recurrent depressive disorder [12]. In patients with anxiety/depression, the antioxidant defense (consisting of enzymes like superoxide dismutase (SOD), catalase (CAT) and nitric oxide synthase (NOS)) is impaired. Immune-inflammatory response is influencing factors for recurrent depressive disorder [12]. In patients with anxiety/depression, the occurrence of chronic inflammatory reactions involving inflammatory signaling pathways and proinflammatory cytokines (e.g. TNF-α, IL-6, IL-1β and IFN-γ) [13,14].A possible link between inflammation and depression was found through the activation of the indoleamine-2,3-dioxygenase (IDO)enzyme. IDO can be activated by pro-inflammatory cytokines and inhibitL-tryptophan (Trp) conversion into serotonin (5-HT) [15,16].Most anti-anxiety/depression drugs act through increasing the levels of 5-HT [17]. Accordingly, therapeutics capable of anti-inflammatory and anti-oxidant likely combated depression and anxiety.

This study aimed to investigate the potential of a formula (40%red pomegranate fruit extract, 34% JB-1, 25% magnesium gluconate and 1% vanillin) to reverse anxiety/depression state and relieve hippocampus neuron injury in mice. Due to the significant effects of the RPFE formula against anxiety/depression, the imparting antiinflammatory, anti-oxidation and neuroprotection properties, which is related to 5-HT synthesis in hippocampus, was further studied.

2. Materials and methods

2.1 Material and chemicals

Red pomegranate fruit extract (SNT181023) was purchased from Xi’an sinuote Bio-Tech Co., Ltd. (Xi’an, China). JB-1 (≥ 5 × 1010CFU)was purchased from Infinitus, (China) Co., Ltd. (Jiangmen, China).Magnesium gluconate (Food grade, 98%) was purchased from Zhengzhou Ruipu Biological Engineering Co., Ltd. (Zhengzhou,China). Vanillin (≥ 98%) was purchased from Norwegian Borregaard(Norway). The ingredients of the formula containing red pomegranate fruit extract are shown in Table 1. Corticosterone was purchased from Beijing Solarbio Science & Technology Co., Ltd. (Beijing,China). Fluoxetine hydrochloride (98%) was purchased from Maibosi Biomedical Co., Ltd., Suzhou, China. Other reagents and chemicals used were purchased from CapitalBio Corporation (Shanghai, China)and of at least analytical grade.

Table 1The ingredients and proportions of the RPFE formula.

2.2 Animals

Healthy male C57BL/6 mice ((18 ± 2) g; aged 3 weeks) were obtained from Changzhou Cavens Laboratory Animal Co., Ltd.(Changzhou, Jiangsu, China). The animals were maintained (five animals per cage (20 × 215 × 170 mm)) under standard conditions: a 12 h light/12 h dark cycle (lights on 07:30-19:30); (55 ± 5)% relative humidity; (25 ± 2) °C; normal rodent chow and waterad libitum. The mice were allowed to acclimatize for 7 days, before animal modeling,administration with the formula, and behavioral experiments during the light phase of the light/dark cycle.

The animal experiments were approved by the Committee for the Care and Use of Laboratory Animals in the South China Agricultural University [IACUC No. 2018D047], and all experimental procedures were performed in compliance with the Author Guidelines on Animal Ethics and Welfare for Veterinary Journals published by the International Association of Veterinary Editors for the protection of animals used for scientific purpose.

2.3 Animals model and treatment

This study used chronic restraint stress (CRS) combined with subcutaneous injection of corticosterone (CORT) to establish a C57BL/6 mouse model of anxiety/depression-like state as the method of Zhu et al. [18]. At the modeling stage (21 days), mice were randomly assigned to three experimental groups and weighed every week. The normal control (NC,n= 10): mice were fasted (6 h/day,9:00-15:00); vehicle control (VG,n= 10): mice were subjected to CRS (6 h/day, 9:00-15:00) and subcutaneous injection with saline in a volume of 0.02 mL/g body weight; model control (n= 50): mice were subjected to CRS (6 h/day, 9:00-15:00) and subcutaneous injection with CORT (30 mg/kg body weight/day, suspended in the physiological saline containing 0.1% dimethyl sulfoxide (DMSO) and 0.1% Tween-80) in a volume of 0.02 mL/g body weight.

At the treatment stage (which followed the 21-day modeling stage), the 50 model control mice with anxiety/depression-like behaviors were randomly assigned to five groups (n = 10 per group)(MC, PC, red pomegranate fruit extract (RPFE) formula at a high dose (RPFEH), RPFE formula at a medium dose (RPFEM), and RPFE formula at a low dose (RPFEL)). The NC (treated with saline),VG (treated with saline), PC (treated with fluoxetine hydrochloride,18 mg/kg·day), MC (treated with saline), RPFEH (treated with RPFE formula at a high dose: 2.0 mg/g·day), RPFEM (treated with RPFE formula at a medium dose: 1.5 mg/g·day), RPFEL (treated with RPFE formula at a low dose: 1.0 mg/g·day). All therapeutic agents were dissolved in the above-mentioned saline (in a volume of 10 mL/kg·day per mouse) and gavaged once daily to mice for four weeks.

2.4 Behavioral assessments

After modeling and at the end of 4-week administration, all mice were subjected to the animal behavior experiments during the day. The movements/behaviors of the animals were tracked and recorded using a digital video camera (Shanghai Bio-will Co., Ltd.Shanghai, China). A calm and consistent environment was maintained throughout all behavioral tests to obtain accurate results. Fresh water was used for each mouse, and the cylinder was also cleaned between trials in forced swimming test (FST). The box and maze were thoroughly cleaned with 70% aqueous ethanol between tests, to eliminate the influence of the odors of urine and feces left from the previous test.

2.4.1 FST

In a FST test, mice were forced to swim for 6 min, with the parameters set as described previously with slight modification[19]. The mouse was placed into a glass cylinder (height, 25 cm tall;diameter, 10 cm; filled with water up to 15 cm; temperature controlled at (24 ± 1) °C). After a 6-min pre-test, the mouse was removed and dried before being placed back into its home cage. After 24 h, the mouse was placed into the cylinder again for repeating the 6-min test.The movements/behaviors of the animals were tracked and recorded using a digital video camera, and the total duration of immobility (in seconds) was measured. Immobility was defined as the absence of motion of the whole body, with the exception for small and gentle movements (such as mild paddling with one foot without struggling)necessary to keep the animal’s head above the water.

2.4.2 Open- field test (OFT)

Animals were individually placed in the far right corner of an open box (40 cm length × 40 cm width × 40 cm height). First,the animal was gently placed in the far right corner of the box and allowed to adapt for 15 min. After one day of the adaptation, each mouse was subjected to a 10-min test: it was placed again in the far right corner of the box and allowed to move freely. All the behaviors of the mice were recorded using a digital video camera.

2.4.3 Elevated plus-maze test (EPM)

The EPM test was conducted using a maze composed of a crossshaped platform with two open arms (30 cm length × 5 cm width)and two closed arms (30 cm length × 5 cm width × 15 cm depth)extended from a common central zone (5 cm length × 5 cm width).The whole maze was elevated to a height of 80 cm off the ground.Each mouse was individually set onto the center of the maze facing one of the opened arms. All the behaviors of the mice were recorded with a digital video camera for 5 min (to analyze the number of entries and time spent on the open arm), according to the method of Walf et al. [20].

2.5 Biochemical analyses for the mice examined

At the end of behavioral tests, the mice were deeply anesthetized with chloral hydrate. The blood from the eyeball was collected into a 1.5 mL centrifuge tube, before the mouse was sacrificed the blood cells in the collected blood were immediately lysed by a RS-120S ultrasonic cell disrupter system (BRANSON, American), and the blood were kept at (25 ± 1) °C for 30 min and centrifuged at 3 000 × g and 4 °C for 15 min to collect the supernatant (serum)using a 5424R centrifuge (Eppendorf, Germany). The supernatants(sera) were collected and stored at -80 °C until further analysis. In the meantime, the brains of the mice were rapidly removed from the skull. The hippocampal tissues were quickly removed and placed on ice, homogenized with ice-cold phosphate buffer saline (PBS,pH 7.4) using a homogenizer (JXFSTPRP-24, Shanghai Jingxin Industrial Development Co., Ltd. Shanghai, China). The homogenized samples were then centrifuge at 4 °C and 3 000 × g for 20 min to collect the supernatants (sera) using the above-mentioned centrifuge.The supernatants (from the hippocampal tissues) were collected and stored at -80 °C until use. The levels of NF-κB, TNF-α, IL-6,IL-1β and IFN-γ in serum were analyzed using the enzyme-linked immunosorbent assay (ELISA) along with the colorimetric test,according to the manufacturer’s instructions. The total activity of CAT, SOD, NOS, IDO, tryptophan hydroxylase (TPH) and the level of MDA, 5-HT in the hippocampal were measured using the ELISA.The levels of serum CRH, ACTH, CORT were measured using ELISA and chemical colorimetry according to the manufacturer’s instructions [21]. All the ELISA kits were purchased from Shanghai Jianglai Biological Technology Co., Ltd. (Shanghai China).

2.6 Histopathological examinations of hippocampal specimens using hematoxylin and eosin (H&E) staining

Freshly collected hippocampal specimens were fixed with 4%neutral formaldehyde fixative for 24 h, embedded in paraffin and cooled at -20 °C. Once the wax was solidified, the block embedded in paraffin wax was removed, trimmed, and cut into 5 μm-thick sections.These tissue sections were stained with H & E and examined under an upright optical microscope (Nikon Eclipse E100, Nikon, Japan) and imaging system (Nikon DS-U3, Nikon, Japan).

2.7 Data analysis

All experimental results were expressed as “mean ± standard deviation” for reporting, and each experiment was repeated at least 3 times. Data were analyzed using SPSS 16.0 (SPSS Inc., Chicago, IL,U.S.A.) statistical software. Statistical significance was set atP＜ 0.05.

3. Results and discussion

3.1 Effects on the body weights of mice

There were no significant differences (P＞ 0.05) in initial body weight ((19.76 ± 0.27) g) among the 7 experimental groups of mice prior to CRS-CORT induction (Table 2). The post-model(0-3 weeks) body weights of the CRS-CORT induced model mice were significantly lower (P＜ 0.05) than those of the NC and VG groups of mice, but the body weights were insignificantly (P＞ 0.05)different within the CRS-CORT induced model group. The body weights of the VG group were significantly lower (P＜ 0.05) than those of the NC mice. After the 4-week chronic treatment, the body weights of all the groups increased. The body weights of the RPFE formula, PC, NC and VG groups were significantly (P＜ 0.05)higher, compared with those of the MC (model control) mice.The body weights of the MC and PC groups in the first week of the administration period (5th week) were significantly lower (P＜ 0.05),compared with those of the NC, VG, RPFEH and RPFEM groups.There were no significant (P＞ 0.05) differences in body weight among the PC, RPFEH, RPFEM and RPFEL groups from the second week of the administration period (6th week).

Table 2The body weights (g, n = 10) of different groups of mice at different time points.

3.2 Behavioral tests

After the 4-week administration, the percentage for the time spent on open arm of the NC mice (9.82%) was significantly(P＜ 0.05) higher than those of the other groups, with the MC mice having the lowest values (2.31%), and the PC (6.98%) and RPFEH(6.54%) mice having significantly (P＜ 0.05) higher values than the VG (3.75%), RPFEM (4.41%) and RPFEL (3.19%) mice (Fig. 1A).The percentage of the time spent in the center for the NC (10.11%)and VG (7.45%) mice remained the highest (P＜ 0.05) (Fig. 1B).However, a remarkable increase was observed in the percentage of the time spent in the center for the PC mice (5.39%), RPFEH mice(4.52%) and RPFEM mice (3.38%), with no change before and after the administration period in the percentage of the time spent in the center for the RPFEL mice (1.52%). A significant decrease (P＜ 0.05)was detected for the MC mice after the 4-week administration (down to 0.43%, which made this group of mice have a lower percentage of the time spent in the center as compared with that (4.52%)for the RPFEH mice). After the 4-week administration, the NC mice did not exhibit anxiety-like behaviors based on the detected movement tracks and maximum depth entered into the open arms in the EPM test (Fig. 1B). The MC mice had fewer movements in the closed arms and no movement in the open arms, and tended to stay in the corner of a closed arm were observed (which indicated significant anxiety-like behaviors). The VG, PC, and RPFE mice showed significantly more movements than the MC mice, although the maximum depth in the open arms and the number of open arm entries of these groups varied. Fig. 1C shows the total movement tracks in OFT (within 10 min): The MC mice mostly stayed far from the center (around the edges and corners), the RPFEL group exhibited slightly more movements towards the center, while the PC, RPFEH and RPFEM showed significantly (P＜ 0.05)more movements including access to the center. Since EPM and OFT assess anxiety-related behaviors and locomotor activity,respectively, although anxiolytic effect and locomotor increment are difficult to differentiate [22]. Accordingly, RPFE was effective in alleviating anxiety-like behaviors in mice.

The immobility time of the CRS-CORT-treated mice was significantly (P＜ 0.05) shortened after the 4-week administration with RPFEH, RPFEM, RPFEL or fluoxetine (PC), compared with the MC mice (Fig. 1A). The difference in the mean immobility time between the RPFEH group and the NC group was statistically insignificant(P＞ 0.05). Therefore, the PC and RPFE formula could alleviate anxiety/depression-like behaviors after the 4-week treatment. Fig. 1D shows the total movement tracks in FST. The MC mice mostly stayed around the edges with fewer movements, suggesting significant depression-like behaviors. The PC mice and the three RPFE groups exhibited significantly (P＜ 0.05) more movements including access to the center. Thus, the RPFE formula could counteract the prolongation of the immobility time caused by CRS-CORT, thereby alleviating effectively depression-like behaviors in mice.

3.3 Effects of RPFE formula on the antioxidant biomarkers in the hippocampus

Anxiety/depression could impair antioxidant defense including aberrations of antioxidant enzymes activities and O&NS pathways [13]. In this study, the MC mice had significantly higher(P＜ 0.05) MDA level and activities of SOD, CAT and NOS(Table 3). Compared with the MC group, the NC, VG, PC, RPFEH,RPFEM and RRPFEL groups had significantly (P＜ 0.05) lower MDA concentration, lower SOD and NOS activities, but statistically the same (P＞ 0.05) CAT activity in the hippocampus. There were no significant differences (P＞ 0.05) in MDA concentration as well as SOD, CAT and NOS activities in the hippocampus among the PC, RPFEH, RPFEM and RPFEL groups. The three RPFE groups exhibited the same changing trends in MDA level and activities of SOD, CAT and NOS in the hippocampus, compared with the PC group. The increases in MDA level and activities of antioxidant enzymes (SOD, CAT, NOS) indicated that ROS and free radicals caused the damage of the antioxidant defense system. After the 4-week treatment with fluoxetine hydrochloride or RPFE formula,the increased MDA level and increased activities of SOD, CAT and NOS in response to CRS-CORT during the modeling period were all normalized. Previous human studies reported that the depressed patients had significantly higher activity levels of CAT and SOD in the sera as compared to healthy volunteers [23]. So, the increased activities of antioxidant enzymes (SOD, CAT, NOS) responding to CRS-CORT exposure reflected the up-regulation of the internal antioxidant defense system in mice against the increased ROS and free radicals caused by anxiety/depression [13]. The treatments with antidepressants were previously found capable of normalizing the increased levels of antioxidant enzymes in depressed patients [24].Accordingly, the treatment with the RPFE formula may exert antianxiety and/or anti-depression effects at least partially through an antioxidant action.

Fig. 1 (A) The effects of RPFE formula on the behaviors of mice in the open- field, elevated plus-maze, and forced swimming tests. Different letters indicate significant differences (P ＜ 0.05). (B) The total movement tracked in EPM test: the red lines were the recorded motion tracks/routes of mice. The arms in the vertical direction were the closed arms, and those in the horizontal direction were the open arms. (C) The total movement tracked in OFT: the red dots indicate where the mice started the test, with the black dots showing where the mice stopped after the 10-min test, and the green lines were the recorded motion tracks/routes of mice. (D) The total movement tracked in FST: the red dots indicate where the mice started the test, with the black dots showing where the mice stopped after the 10-min test, and the green lines were the recorded motion tracks/routes of mice.

Table 3Effects of RPFE on MDA levels and antioxidant enzyme activity (n = 10).

3.4 Effects of RPFE formula on inflammatory cytokines

The MC mice had higher levels of serum inflammatory cytokines(NF-κB, TNF-α, IL-6, IL-1β and IFN-γ), compared with the other groups (Table 4). Except for the RPFEL mice, the levels of NF-κB,IL-1β and IFN-γ in the MC mice were significantly (P＜ 0.05) higher than those of the other groups. The levels of IL-6 in all the RPFE (H,M, L) mice were significantly (P＜ 0.05) lower than those of the other groups, with the RPFEH having the lowest level. The levels of NF-κB,TNF-α and IFN-γ in RPFE (H, M, L) mice were significantly(P＜ 0.05) higher than those of the NC mice, but significantly(P＜ 0.05) lower than those of the MC mice. The levels of NF-κB,TNF-α, IL-1β and IFN-γ in the PC mice were significantly (P＜ 0.05)lower than those of the RPFE (H, M, L) mice. These results indicated that long-term exposure to CRS-CORT during the modeling period involved inflammatory response and caused increases in levels of pro-inflammatory cytokines such as TNF-α, IL-6, IL-1β and IFN-γ)[18]. The expression of a variety of innate immune genes and proteins(including cytokines, chemokines, enzymes and other inflammatory molecules such as TNF-α, IL-1β, IL-6, IFN-γ) was found to increase in post-mortem brain samples obtained from suicide victims with depression [25]. Peripheral blood IL-1β, IL-6 and TNF-α were reported as the most reliable biomarkers of inflammation in patients with depression [26], and their expression levels can be normalized after a successful treatment with an antidepressant [27]. Accordingly,the RPFE formula has the ability to regulate the normalization of the inflammatory cytokines levels in anxious/depressed mice caused by CRS-CORT, and such an ability likely increases the effect of RPFE to regulate inflammatory cytokines normalization was increased in a dose-dependent manner.

Table 4Effects of RPFE on the levels of NF-κB, TNF-α, IL-6, IL-1β and IFN-γ(n = 10).

3.5 Effects of RPFE formula on HPA axis regulation

Anxiety/depression could incite HPA axis dysregulation including the higher levels of serum CRH, ACTH and CORT [28]. In this study, the MC mice had significantly higher (P＜ 0.05) CRH and ACTH levels and significantly lower (P＜ 0.05) CORT level (Fig. 2).Compared with the MC group, the NC, VG, PC, RPFEH, RPFEM and RRFEL groups had significantly lower (P＜ 0.05) CRH and ACTH levels, higher CORT level. Compared with the NC group, the VG,RPFEH, RPFEM and RRFEL groups had significantly (P＜ 0.05)higher CRH and ACTH levels, lower CORT level. There were no significant (P＞ 0.05) differences in CRH level between NC and PC groups. The three RPFE groups exhibited the same changing trends in CRH, ACTH and CORT levels compared with the PC group. But the PC mice had significantly higher (P＜ 0.05) CORT level and significantly lower (P＜ 0.05) CRH and ACTH levels compared with the three RPFE groups. The 3 RPFE groups can increase CORT level and reduce CRH and ACTH levels, with the RPFE high dose being the most effective. The increase in CORT level and reduces in CRH and ACTH levels indicated that HPA axis secretion tends to normalize.

Fig. 2 The effects of RPFE on plasma CRH, ACTH and CORT levels.Different letters indicate significant differences (P ＜ 0.05).

After the 4-week treatment with fluoxetine hydrochloride or RPFE formula, the increased CRH and ACTH levels and reduced CORT level in response to CRS-CORT during the modeling period were all normalized. Atrophy of the adrenal cortex leads to insufficient secretion of endogenous CORT, which will lead to adrenal insufficiency when the supply of exogenous CORT is suddenly stopped [29]. Over-expression of CORT reduces the expression of glucocorticoid receptors in the hippocampus and impairs glucocorticoid-mediated negative feedback inhibition of the HPA axis destroy the negative feedback regulation of the HPA axis, whilst acute exposure to high levels of glucocorticoids in the hippocampus can exhibit negative feedback regulation of the HPA axis [30,31]. Therefore, this was why MC mice have higher plasma CRH and ACTH levels and lower CORT level compared with the other groups of mice. Since there is evidence that Mg2+modulates the HPA axis and Mg2+supplementation reduces the anxiety/depression-related behavior of mice [32]. Accordingly,the treatment with the RPFE formula may exert anti-anxiety and/or anti-depression effects at least partially through Mg2+supplementation and HPA axis regulation.

3.6 Effects of RPFE formula on 5-HT level in hippocampus

After the 4-week treatment of this study, the NC mice had normal 5-HT level and IDO, TPH activities in hippocampus (Table 5). The MC mice had lower 5-HT level and TPH activity compared with the other groups. But the MC mice had significantly higher (P＜ 0.05)IDO activity compared with the other groups. The impact of RPFE (H,M and L) and PC groups on 5-HT levels and TPH activities exhibited the same trend, but PC group was significant higher (P＞ 0.05) than RPFE (H, M and L) groups. No significant difference was found between PC and RPFEH, RPFEM on IDO activity. The level of 5-HT and activity of TPH in RPFEH and RPFEM groups were significant higher (P＞ 0.05) than RPFEL group. No significant difference was found between RPFEH and RPFEM on 5-HT level and TPH activity. The IDO activity of RPFEH group was significant lower(P＞ 0.05) compared with RPFEM and RPFEL groups. No significant difference was found between RPFEM and RPFEL on IDO activity.IDO (activated by pro-inflammatory cytokines in extrahepatic tissue)is important rate-limiting enzymes in kynurenic (KYN) synthesis and catalyzes the conversion of Trp to KYN and then to QUIN (an excitotoxic metabolite) [16]. TPH is another important rate-limiting enzyme in 5-HT synthesis and catalyzes the conversion of Trp to 5-hydroxytryptophan (5-HTP) and then to 5-HT [33]. The decreased concentration of 5-HT and increased IDO activity in the brain is a contributor to the etiology of anxiety/depression [34]. Our result indicated that after ingestion of RPFE diets indeed enhanced 5-HT level in hippocampus, with the RPFEH has better effective. Therefore,RPFE can significantly and more effectively reduce activity of IDO caused by CRS combined with CORT injection in mice viaantiinflammatory pathways.

Table 5Effects of RPFE on 5-HT level in hippocampus (n = 10).

Fig. 3 The histopathological alterations (shown by H & E staining; magnification 400 ×) in hippocampal CA3 regions caused by RPFE. a-g represent NC, VG,MC, PC, RPFEH, RPFEM, RPFEL, respectively.

3.7 Histological studies

Dendritic spines the in hippocampal CA3 region are major sites for storing synaptic strength, receiving input from a single axon at the synapse, processing electrical signals and information in the brain [35].Loss of dendritic spines and damage of hippocampal CA3 pyramidal neurons were found in chronically stressed animals and depressed subjects [36]. In this study, the pyramidal cells in the hippocampal CA3 region of the NC mice were closely arranged and shaped like an ellipse with a normal structure (Fig. 3). The MC mice exhibited more severe hippocampal atrophy, shape deformation, cellular loss, and dysregulation of hippocampal neurogenesis. In particular, the CA3 neurons of the MC mice contained a reduced number of cells, with increased cytoplasmic vacuolation, nuclear pyknosis, cell membrane shrinkage, irregular cell morphology (triangle-like shape) and obscure cell boundaries. Histopathological changes in the hippocampus of the anxious/depressed mice were significantly (P ＜ 0.05) alleviated after the treatment with the RPFE formula or fluoxetine hydrochloride(the hippocampus of the RPFE and PC group was recovered to the state close to that of the NC group). Thus, the RPFE formula could effectively ameliorate pathological changes in the hippocampal neurons of the mice with anxiety/depression.

4. Conclusion

The findings of this study help justify the traditional use of red pomegranate and vanillin for the treatment of anxiety/depression.The RPFE formula containing the red pomegranate fruit extract(40%, m/m), JB-1 (34%, m/m), magnesium gluconate (25%, m/m)and vanillin (1%, m/m) was effective in relieving anxiety/depressionlike symptoms in mice. Significant reversing effects of the RPFE formula were found on the following parameters of anxious/depressed mice: body weight, inflammatory cytokines, HPA axis regulation,5-HT synthesis, activities of IDO, TPH and antioxidant enzymes,and hippocampal neurons. Neurons were protected by RPFE formula through activating antioxidant enzymes, thus alleviating the MDA-induced damage. The 5-HT synthesis was increased by inhibiting the IDO activity via RPFE formula anti-inflammatory effect. HPA axis normalization was considered to regulate by Mg2+, then reverse anxiety and depression symptoms. Based on the demonstrated antianxiety, anti-depression, anti-inflammatory, anti-oxidation and HPA axis regulation effects of the RPFE formula, the RPFE formula might be an effective therapeutic product to help treat anxiety/depression.

conflicts of interest

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Acknowledgment

The authors are grateful for the financial support from National Natural Science Foundation of China (No. 31201416) and Science and Technology Research Funds of Guangdong Province(No.2017A010105002).