Amelioration of nonylphenol-induced anxiety/depression-like behaviors in male rats using green tea and Zn-Se tea interventions

doi:10.21203/rs.3.rs-2049369/v1

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Amelioration of nonylphenol-induced anxiety/depression-like behaviors in male rats using green tea and Zn-Se tea interventions

https://doi.org/10.21203/rs.3.rs-2049369/v1

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We investigated the effects of nonylphenol (NP) on anxiety/depression-like behaviors and alleviation via green tea and zinc selenium (Zn-Se) tea interventions in rats. Forty male specific-pathogen free (SPF) Sprague-Dawley (SD) male rats were randomly divided into four groups (n = 10 per group): control group (C); NP group (40 mg/kg NP); green tea group (40 mg/kg NP + 0.2 g/mL GT group) and Zn-Se tea group (40 mg/kg NP + 0.2 g/mL ZST group). Following tea intervention, compared with the NP group, the residence time in the light-dark box test decreased, and the number of entries into the closed arm in the elevated plus maze test in the tea-treated group was significantly reduced. The sucrose preference index in the sucrose preference test increased, and the immobility time in the forced swimming test decreased. The effect of Zn-Se tea was better than that of green tea. The damage to the hippocampal tissues in the group treated with tea was less than in the NP group. The cellular arrangement was tighter with degeneration, deep staining, and pyknotic nerve cells were visible. The nuclei of the NP group were atrophied, and the cells were sparsely arranged. Compared with the NP group, corticosterone levels were decreased in the NP + Zn-Se tea group. Chronic NP exposure induced anxiety/depression-like behaviors in rats. Green tea effectively reduced the damage to the hippocampal and prefrontal cortex induced by NP. The effects of Zn-Se tea were slightly more optimal than those of conventional green tea.

Nonylphenol

zinc selenium (Zn-Se) tea

anxiety/depression-like behaviors

BDNF

Exposure to nonylphenol (NP) induces anxiety/depression-like behaviors ^[1–2]. Oral perfusion of NP alone or in combination can result in anxiety in rats, and aggressiveness is enhanced during gavage accompanied by mental sluggishness with time ^[3]. You et al ^[4] demonstrated that exposure to NP by pregnant rats resulted in defective social behavior in offspring. Yang et al ^[5] reported that NP contamination led to varying degrees of dehairing, and decreased activity and appetite. In addition, L-theanine (L-THE) combined with caffeine promotes antioxidant and anti-inflammatory activity in the brain, which might reduce the risk of cognitive disorder. Supplementation of L-THE (200–400 mg/day) might contribute to the reduction of stress and anxiety in individuals exposed to stressful conditions ^[6]. The combination of L-THE, polyphenols and polyphenol metabolites can potentially reduce the risk of depression via multiple signaling pathways ^[7]. Selenium (Se) is an essential trace element in the human body, and Se deficiency is closely related to the development of anxiety and depression ^[8]. Adequate intake of zinc (Zn) and Se prevents anxiety/depression, which is proportional to the levels of Zn and Se in the human body ^[9–10]. Previous studies have demonstrated stronger anti-oxidant and anti-aging effects of green tea containing abundant Zn and Se compared with conventional green tea ^[11–12]. Additionally, the Zn-Se tea of Guizhou can effectively ameliorate disorders associated with glucose and lipid metabolism, promotes fat metabolism, and antagonizes the toxic effects of NP in the body ^[13]. Nevertheless, the ability of Zn-Se tea to ameliorate NP-induced anxiety/depression behaviors remains unclear.

It has been demonstrated that the antioxidative Zn and Se play a vital role in improving cognition and resisting oxidative stress, which are associated with mental deterioration ^[14]. Indeed, Zn and Se may have preventive and protective effects on the occurrence and progression of Alzheimer’s disease ^[15]. Xu et al ^[16] demonstrated that Zn and Se can change the levels of monoamine oxidase (MAO), acetylcholinesterase (AChE), and malondialdehyde (MDA) in the brain, and play a regulatory role in Alzheimer’s disease. Also, the small molecule organic Se compounds can protect hippocampal neurons and increase the learning and memory abilities of rats ^[17]. Therefore, our study investigated the role of green tea and Zn-Se tea in alleviating anxiety/depression-like behaviors in rats induced by long-term NP exposure via continuous NP gavage to 40 Sprague-Dawley (SD) male rats at a dose of 40 mg/kg/d for 6 months, followed by 1 g/kg/d of green tea and Zn-Se tea after 3 months. Our study provides a standard of reference for the prevention and treatment of anxiety and depression using green tea and Zn-Se tea.

2.1 Reagents and equipments

The nonylphenol (99% > purity, Product Code: BT5655) was purchased from the Shandong Xiya Chemical Industry Co., Ltd. (Shandong, China). Rat corticosterone ELISA kit, rat BDNF ELISA kit were all purchased from Wuhan Cloud-Clone Corp. (Wuhan, China). Hematoxylin and eosin staining kit (Code: G1018) were purchased from the Wuhan Guge biological Co., Ltd. (Hubei, China). HP-1100 High Performance Liquid Chromatography (HPLC) was purchased from the Agilent Technologies (Palo Alto, CA, USA). SuperMaze animal behavior video analysis system was purchased from Shanghai Xinruan Information Technology Co., Ltd. (Shanghai, China). Optical microscope was purchased from Olympus (Tokyo, Japan). All other chemicals were commercially available.

2.2 NP contamination dose

As reported, the allowable daily intake of NP in humans is 5 µg/kg•bw/day. According to the security coefficient (human:rat = 1: 100), the tolerable daily NP intake by rats was calculated to be 0.5 mg /kg•bw/day. Accordingly, the doses of 0.4 mg/kg•bw/day, 4 mg/kg•bw/day, and 40 mg/kg•bw/day were used for NP gavage. Previous studies reported that long-term NP gavage at 40 mg/kg•bw/day can cause anxiety/depression-like behaviors ^{[1, 18]}. Therefore, the dose of 40 mg/kg•bw/day was used for NP gavage in this study.

2.3 Preparation of tea leaves and tea

The tea leaf dose for intervention was 1 g/kg/d and the gavage volume was 5 mL/kg. As previously reported, the daily amount of tea consumed by the general population is 3.61 g, with an average number of 3.86 cups of tea consumed per day ^[19]. In the current study, the concentration of tea used was 0.2 g/mL, which was equivalent to 18.05 mL/d of strong tea. Tea volume was determined by dividing the amount of tea consumed by the concentration of tea (3.61 g / 0.20 g/mL, Table 1).

Similar amounts of Zn-Se tea and green tea were weighed and added to three volumes of boiling distilled water. After boiling the water three times in a row for 15 min, the liquids were concentrated to 0.2 g/mL and stored in a refrigerator at 4°C for later use.

Table 1

Zn and Se contents in tea.
Group	Place of Origin	Manufacturer	Zn content	Se content
Green tea	Fenggang county of Guizhou	Guizhou Flavor tea Co., Ltd (Guizhou, China)	40.0-100.0	0.05–2.57
Zn-Se tea	Fenggang county of Guizhou	Guizhou Wanhuyuan Zinc Selenium Tea Co., Ltd (Guizhou, China)	55.4-103.2	1.38–2.03

2.4 Animal groupings and treatment

Forty male SPF SD rats aged four weeks and weighing 110–120 g were used in this study. The rats were purchased from Changsha Tianqin Biotechnology Co., Ltd. (License No: SCXK2014-0011, Changsha, China). The rats were randomly divided into four groups (n = 10 per group) after adaptive feeding for 1 week: control group (C) fed with 5 mL/kg corn oil daily; NP group (NP) treated with 40 mg/kg NP daily; green tea group exposed to 0.2 g/mL (NP + GT group); and 0.2 g/mL Zn-Se tea group (NP + ZST group). NP at 40 mg/kg/d was administered to the tea groups for 3 months, followed by NP + green tea and NP + Zn-Se tea for 3 months. Rats were weighed at 8:00 AM and exposed to NP by gavage with a volume of 5 mL/kg/d.

The rats were housed in a clean animal room at a temperature of 24 ± 2°C, relative humidity of 45–55%, under artificial lighting (12 h light/12 h dark). They were fed a standard maintenance diet of pellet feed (License No: SCXK (Chuan) 2015-01). Animal experiments have been approved by the Ethics Committee of Zunyi Medical University [No.: Lun Shen (2016) 2–068]. A flowchart depicting exposure protocols for the rats and the duration of exposure is presented in Fig. 1.

2.4.1 Measurement of NP in hippocampal tissues

NP levels were measured in 100 mg of hippocampal tissues in a glass tube. Following addition of 2 mL of n-hexane and diethyl ether (n-hexane:diethyl ether = 7:3), the mixture was centrifuged at 3000 r/min for 10 min. The supernatant was then transferred to another clean tube and evaporated in a water bath at 50°C in a fume hood. Next, 0.5 mL of acetonitrile was used for elution. The sample was transferred to a sample bottle for HPLC analysis under the following mobile phase conditions: acetonitrile: glacial acetic acid, 85:15; excitation wavelength, 275 nm; emission wavelength, 312 nm; column temperature, 40°C; injection volume, 10 µL; and flow rate, 1.0 mL/min. The NP peak time was 3.4 min ^[20].

2.4.2 Ethology

2.4.2.1 Detection of anxiety-like behaviors in rats using light-dark box test

The rat light-dark box was composed of a polypropylene animal box (44×21×21cm), divided equally between light and dark zones (50% each). A small hole between the light and the dark zones of the box enabled free movement of the rats. The duration of activity in the light and dark areas of the box within 5 min was recorded. The symptoms of anxiety were determined by the total distance traveled in the box and were proportional to the dark zone residence time ^[21].

2.4.2.2 Detection of anxiety-like behaviors in rats using elevated plus maze test

The maze device was composed of two open arms (50×10×50cm) and two closed arms (50×10×50cm). The duration of rat entry and stay in the closed arm within 300 s was recorded. The entry time and duration of stay in the closed arm were proportional to the severity of anxiety ^[22].

2.4.2.3 Detection of anxiety-like behaviors in rats via open-field test

The open-field test was used to evaluate locomotor activity and anxiety and depression-like behaviors of rats. The open-field behavior observation box was 100 cm long, 100 cm wide and 40 cm high ^[23]. The activity of rats in 5 min was recorded, including the mean speed of movement and immobility time in the open field test.

2.4.2.4 Detection of depression-like behaviors in rats via sucrose preference test

During the first 24 hours, each cage was supplied with two bottles of 1% (w/v) sucrose solution. A bottle was replaced with water at 24 h. After adaptation, fasting was performed for 12 h, and detection was initiated. For detection, each cage was equipped with two bottles, one containing 500 mL pure water, and another bottle with 500 mL of a 1% (w/v) sucrose solution. After 12 hours, the two bottles were removed, and the consumption of sugar solution and pure water was recorded to calculate the sucrose preference index (sucrose preference index (%) = sugar water consumption / (sugar water consumption + pure water consumption) × 100) ^[24].

2.4.2.5 Detection of depression-like behaviors in rats via forced swimming test

Transparent cylindrical plexiglass tubes measuring 40 cm in height, a radius of 30 cm, water depth of approximately 30 cm, and a water temperature of 23 ± 2°C were used. Following the immersion of rats in the water, the immobility time within 5 min was recorded ^[25].

2.4.3 Pathological examination of hippocampal and prefrontal cortex tissues

After the rats were anesthetized with urethane (0.5 mL / 100 g), the hippocampus and prefrontal cortex were removed, and then fixed with 4% paraformaldehyde, embedded, sectioned, deparaffinized, stained by hematoxylin-eosin, and photographed. Next, morphological changes in hippocampal neurons and prefrontal cortex cell were observed under an optical microscope. Three fields of view were selected from each slice for counting cells under a 400 microscope, with 2 slices per group ^[26].

2.4.4 Measurement of brain-derived neurotrophic factor (BDNF) in hippocampal tissues by ELISA

Phosphate-buffered saline (PBS, PH7.4) was added to 50 mg of hippocampal tissues, which were then thoroughly homogenized. After centrifuging for 20 min at 2,000–3,000 rpm, the supernatant was carefully collected into an Eppendorf tube for subsequent experiments. The experiment was carried out in strict accordance with the instructions provided by the ELISA kit manufacturer ^[27].

2.4.5 Measurement of serum corticosterone by ELISA

The addition of 50 µL standard and 50 µL sample was followed by treatment with 50 µL solution A. The plate was incubated at 37°C for 1 h. After the plate was spun dry and rinsed three times, 100 µL solution B was added, and the plate was then incubated at 37°C for 30 min. Next, the plate was washed 5 times, followed by the addition of 90 µL substrate, and then incubated at 37°C for 10 min. Next, 50 µL stop solution was added, and a microplate reader was immediately used to read the wavelength at 450 nm.

2.5 Statistical analysis

The experimental data are expressed as the mean ± standard deviation (SD,‾X ± S), and SPSS 26.0 software was used for statistical analysis. The comparison of mean values between multiple groups was performed via one-way analysis of variance. Pairwise comparison was performed via LSD and SNK-q test, with significance (α) of the test considered at 0.05.

3.1 Weight variation across different groups

Compared with the control group, the weight of the SD rats increased after NP exposure (F = 4.265, P = 0.01). Intervention with tea led to weight loss in the rats belonging to each group in the following order: control group < NP + Zn-Se tea intervention group < NP + green tea intervention group < NP group (Fig. 2).

3.2 Measurement of NP in hippocampal tissues by HPLC

Compared with the control group, the levels of NP in the hippocampal tissues of the NP group, the green tea intervention group, and the Zn-Se green tea intervention group increased significantly after NP contamination (F = 4.803, P_NP = 0.002, P_GT = 0.004, P_ZST = 0.008), while the concentrations of NP in the hippocampal tissues of the tea group were consistent with those of the NP group (Fig. 3).

3.3 Ethological test

Compared with the control group, the residence time in the light-dark box test after NP exposure was prolonged (P < 0.001). The frequency of entry into the closed arm in the elevated plus maze test in the NP exposure group increased significantly (P_NP = 0.001, P_GT = 0.016). The immobility time was increased in the central square during the open field test (P_NP < 0.001, P_GT = 0.012). The sucrose preference index in the sucrose preference test was reduced (P < 0.001), while the immobility time in the forced swimming test was increased (P < 0.001). However, no significant difference was found between the groups in total distance traveled in the light-dark box and the mean speed of movement in the open-field test. After tea intervention, compared with the NP group, the residence time in the light-dark box was decreased (P_GT = 0.048, P < 0.001), and the frequency of entry into the closed arm of the elevated plus maze test in the group exposed to tea was significantly reduced (P_GT = 0.285, P_ZST = 0.022). In addition, the immobility time was decreased in the central square in the open-field test (P_GT = 0.001, P_ZST < 0.001). The sucrose preference index in the sucrose preference test increased (P_GT = 0.009, P_ZST < 0.001), while the immobility time in the forced swimming test decreased (P_GT = 0.049, P_ZST < 0.001). Further, compared with the green tea group, the residence time in the light-dark box test in the Zn-Se tea group was significantly reduced (F = 12.400, P = 0.045). Fewer entries into the closed arm were detected in the elevated plus maze test (F = 5.805, P = 0.049). Immobility time in the central square in the open field test was significantly decreased (F = 19.186, P = 0.039). The sucrose preference index in the sucrose preference test was significantly elevated (F = 26.430, P = 0.046), while the immobility time in the forced swimming test was significantly decreased (F = 21.913, P = 0.029, Fig. 4).

3.4 Pathology of hippocampal tissues in each rat group

As observed with the microscope, the hippocampal cell nuclei of the control group were complete, round, and large, with rich cytoplasm, while the cellular arrangement in the NP group was disordered and sparse. The damage to the hippocampal tissues in the tea intervention group was less than in the NP group (indicated by the pane), and the cellular arrangement was tighter with degeneration, deep staining, and pyknotic nerve cells were visible (indicated by the arrow, Fig. 5).

3.5 Effects of NP on the number of atrophied nerve cells in hippocampal tissues

Compared with the control group, the number of shrunken cells in the NP group was increased per magnification (P < 0.001). Compared with the NP group, tea intervention led to a decrease in the number of damaged cells in hippocampal tissues, and the number of damaged cells in hippocampal tissues in the NP + Zn-Se tea intervention group was the least (P < 0.001). Compared with the NP + GT group, the number of damaged cells in the hippocampal tissues of rats in the NP + Zn-Se tea group obviously decreased (F = 13.948, P = 0.016, Fig. 6).

3.6 Histopathology of the prefrontal cortex of rats in each group

Hematoxylin and eosin (HE) staining of the prefrontal cortex of rats revealed a clear demarcation of the cytoplasm and nuclear membrane in the neurons of the control group of rats, and the cells were plump. The nuclei of the NP group were shrunken (indicated by the arrow), and the cells were sparsely arranged (indicated by the pane). The cells in rats treated with NP + Zn-Se tea and NP + green tea were slightly reduced in size (Fig. 7).

3.7 Effects of NP on the number of atrophied nerve cells in prefrontal cortex tissues

Compared with the control group, the number of shrunken cells in the NP group was increased under each magnification. The number of pathologically damaged cells in the prefrontal cortex was reduced by tea intervention compared with cells in the NP group, and the least number of cells were damaged in the prefrontal cortex of rats treated with NP + Zn-Se tea. Compared with the NP + green tea group, the number of damaged cells in NP + Zinc-se tea group was significantly reduced (F = 43.635, P < 0.001, Fig. 8).

3.8 Measurement of BDNF levels in hippocampal tissues

Serum BDNF levels in rats with a history of NP exposure were lower than in the control group, and the BDNF level in the NP group decreased significantly (F = 3.037, P = 0.012). Although the results of the two tea groups were not statistically different from the NP group, there was still a slight increase in BDNF level, and the increase in the NP + Zn-Se tea intervention group was more prominent than in the NP + green tea intervention group (Fig. 9).

3.9 Measurement of serum corticosterone

Compared with the control group, the serum corticosterone levels of the NP group were increased significantly (P = 0.008). Compared with the NP group, the corticosterone levels in the sera of rats in the NP + green tea intervention group slightly decreased (P = 0.417) and decreased significantly in the NP + Zn-Se tea intervention group (P = 0.001). Compared with the NP + green tea group, the group treated with NP + Zn-Se tea showed lower levels of serum corticosterine (F = 6.410, P = 0.007, Fig. 10).

In this study, dietary interventions via green tea and Zn-Se tea were investigated for the first time to ameliorate anxiety/depression-like behaviors in rats subjected to long-term NP exposure. The results showed that the weights of NP-exposed rats increased, while the weights of rats treated with tea were less than in the NP group. The weights of rats treated with Zn-Se tea were less than in those exposed to ordinary tea, which may be related to the appetite-regulating Zn content in Zn-Se tea. Green tea effectively reduced the damage to hippocampus and prefrontal cortex induced by NP exposure and hereby ameliorated the anxiety/depression-like behaviors in rats. The effects of intervention with Zn-Se tea were slightly better compared with those of conventional green tea. In this study, NP-induced anxiety/depression-like behaviors in rats treated with green tea and Zn-Se tea were evaluated in terms of weight changes, NP concentrations in the hippocampus, results of anxiety/depression based on ethological tests, hippocampal and prefrontal cortex pathology, levels of neurotrophic factors, and comprehensive analysis of changes in serum corticosteroid levels.

The levels of NP in hippocampal tissues were not significantly reduced by tea intake. The NP levels of rats exposed to NP, green tea, and Zn-Se tea were all higher than those of the control group, suggesting that tea leaf intervention may offset the toxic effect of NP.

In this study, the classical ethological test for anxiety/depression was used to evaluate mental disorders in rats. The light-dark box, the elevated plus-maze, and the open-field tests are classic ethological tests for anxiety evaluation ^[28–29]. NP exposure led to an increase in the residence time in the light-dark box test, the frequency of entry into the closed arm of the elevated plus maze test, while the rat immobility time in the central square in the open field test was reduced, which suggested that the rats lost interest in exploring external objects. While no significant difference was found between the groups in the total distance traveled in the light-dark box, and the mean speed of movement in the open-field test, the loss of interest in the external environment might be attributed to anxiety/depression induced by NP rather than impaired motor function following NP gavage. In addition, the dark zone residence time and the frequency of entry into the closed arms were decreased compared with the NP group. Anxiety-like behavior was more obvious in the ordinary tea group than in the group treated with Zn-Se tea. Sucrose preference and forced swimming are important indicators of depressive behavior in animals. In the current study, Zn-Se tea elevated the sucrose preference index better than green tea, which was decreased by NP. Compared with the NP group, the resting time of forced swimming of rats exposed to green tea and Zn-Se tea was significantly reduced, and the decrease in rats exposed to Zn-Se tea was more obvious than in those exposed to green tea. In summary, green tea and Zn-Se tea decrease the NP-induced anxiety/depression-like behaviors of rats.

The pathology results showed significantly fewer lesions in the hippocampal neurons and the prefrontal cortex of rats treated with tea when compared with NP. Despite fewer cells and irregular arrangement in the Zn-Se tea intervention group, the hippocampus damage was less than in the NP group. Yu et al ^[30] reported that tea leaves had a protective effect on damaged hippocampal tissues. The cytoplasm and nuclear membrane of neurons in the prefrontal cortex of rats in the control group were clearly demarcated. Compared with the control group, the nuclei of neurons in the NP group shrank, and the nuclei in the tea groups shrank slightly. The results demonstrate that consumption of green tea and Zn-Se green tea can improve the nuclear morphology of NP-induced neurons.

BDNF plays an important role in the pathogenesis of mood and anxiety, and decreased BDNF is an important indicator of anxiety ^[31]. The results of this study reveal that long-term NP exposure leads to reduced BDNF levels. Yu et al ^[18] reported that exposure to 4 mg/kg/d NP for three months reduced the BDNF levels in the serum of rats. Tea intervention in the current study led to increased BDNF levels compared with the NP group. Likewise, Kita et al ^[32] demonstrated that intake of tea led to increased BDNF, which further proved that NP exposure reduced BDNF levels, and tea reduced the neurotoxic effects of NP. Increased corticosterone levels in the serum induce depression. Indeed, the intake of Zn and Se can reduce the risk of depression ^[33]. In the current study, the serum corticosterone levels in the NP group were higher than in the control group. Compared with the NP group, the serum corticosterone levels of rats exposed to Zn-Se tea were lower than in rats exposed to green tea, suggesting that tea, especially Zn-Se tea, can mitigate NP-induced increases in serum corticosterone levels. The protective effect of Zn-Se tea on neurological diseases has yet to be elucidated. Studies have shown that the intake of Zn-Se tea improved the antioxidant capacity better than the ordinary green tea^[34]. The antioxidant activity of Se-enriched tea was significantly higher than that of regular tea^[35]. In addition, compared with regular Oolong tea extract, Se-enriched Oolong tea extract exhibited more prominent anti-proliferative effects against human hepatoma HuH-7 cells, mainly due to the synergistic effects of organic selenium and tea polyphenols^[36], which is consistent with the results of this study.

Chronic NP exposure induced anxiety/depression-like behaviors in rats. Intake of green tea effectively reduced the damage to the hippocampal and prefrontal cortex induced by NP, and the effects of intervention with Zn-Se tea were slightly more optimal than those of conventional green tea.

Our results revealed that green tea and Zn-Se tea alleviated anxiety/depression-like behaviors in rats subjected to long-term NP exposure, and the effect of Zn-Se tea was stronger than that of green tea, which provides a rationale for the prevention and treatment of anxiety/depression. Nevertheless, we performed only a qualitative study, suggesting the need for an epidemiological study involving a large population sample. Additionally, characterization of the active ingredients of Zn-Se tea is needed to determine the optimal concentrations required for tea consumption as well as in-depth exploration of the mechanism underlying alleviation of anxiety and depression-like behaviors.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (22166035); Guizhou High-Level Innovative Talent Support Program (GCC[2022]035-1, [2020]6014).

Funding

This work was supported by the National Natural Science Foundation of China (22166035); Guizhou High-Level Innovative Talent Support Program (2022, [2020]6014); 15851 Project Talent in Zunyi municipal government, Guizhou Province (2018(E-262)); Science and Technology Foundation of the Health Commission of Guizhou Province (gzwkj2021-39, gzwkj2021-536).

Authors’ contributions

Jie Xu and Jie Yu designed the study. Yujie Zhang, Jie Xu, Lin Zhu, Shengnan Li, Mizhuan Li, Dayan Tong and Jie Yu analyzed and interpreted the data. Shengnan Li conducted the laboratory work. Shengnan Li, Jie Xu participated in the sample collection. Jie Xu and Yujie Zhang wrote the manuscript, Jie Yu revised the manuscript. All the authors read and approved this paper.

Availability of data and materials

The datasets used and/or analysed during the current study available from the corresponding author on reasonable request.

Competing interests

The authors declare that they have no competing interests.

Ethics approval and consent to participate

Animal experiments have been approved by the Ethics Committee of Zunyi Medical University [No.: Lun Shen (2016) 2-068].

Consent for publication

All the authors read and approved this paper.

Competing interests

The authors declare that they have no competing interests.

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Amelioration of nonylphenol-induced anxiety/depression-like behaviors in male rats using green tea and Zn-Se tea interventions

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Abstract

Figures

1. Introduction

2. Materials And Methods

2.1 Reagents and equipments

2.2 NP contamination dose

2.3 Preparation of tea leaves and tea

2.4 Animal groupings and treatment

2.4.1 Measurement of NP in hippocampal tissues

2.4.2 Ethology

2.4.2.1 Detection of anxiety-like behaviors in rats using light-dark box test

2.4.2.2 Detection of anxiety-like behaviors in rats using elevated plus maze test

2.4.2.3 Detection of anxiety-like behaviors in rats via open-field test

2.4.2.4 Detection of depression-like behaviors in rats via sucrose preference test

2.4.2.5 Detection of depression-like behaviors in rats via forced swimming test

2.4.3 Pathological examination of hippocampal and prefrontal cortex tissues

2.4.4 Measurement of brain-derived neurotrophic factor (BDNF) in hippocampal tissues by ELISA

2.4.5 Measurement of serum corticosterone by ELISA

2.5 Statistical analysis

3. Results

3.1 Weight variation across different groups

3.2 Measurement of NP in hippocampal tissues by HPLC

3.3 Ethological test

3.4 Pathology of hippocampal tissues in each rat group

3.5 Effects of NP on the number of atrophied nerve cells in hippocampal tissues

3.6 Histopathology of the prefrontal cortex of rats in each group

3.7 Effects of NP on the number of atrophied nerve cells in prefrontal cortex tissues

3.8 Measurement of BDNF levels in hippocampal tissues

3.9 Measurement of serum corticosterone

4. Discussion

5. Conclusions

Declarations

References

Additional Declarations

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