**2.6 Sucrose preference test**

*Vitamin D Deficiency*

of depression state is strongly verified by both preclinical and clinical studies [46]. CUMS was made as described previously [47, 48]. The procedure included the exposure to different and unpredictable stress factors that are randomly changed during experimental days [49]. These manipulations are 24 h food deprivation, 24 h water deprivation, wet bedding overnight, titled cage overnight, unpredictable shocks (15 mA, one shocks/20s, 10 s duration, 20 min), 5 min swimming at cold water (4°C), tail hanging, 1 min, clip tail for 1 min, reversal of light/dark cycle [47, 48]. All stress triggers were performed individually and continuously. To prevent habituation and to ensure the unpredictability of the stressors, all stress manipulations randomly made accordingly to experimental scheme, repeated throughout the 4 weeks of CUMS protocol. The control SHAM females were placed in a separate room without any contact with the stressed groups of animals. These rats were maintained as undisturbed animals that are subjected only routine cage cleaning for 4 weeks.

*Timeline of chronic treatment. Female Wistar rats were divided into 6 groups – non-CUMS SHAM rats treated with solvent (control), SHAM rats submitted to CUMS treated with solvent, long-term OVX rats exposed to CUMS given with solvent, fluoxetine as positive control (10.0 mg/kg/day), 17β-E2 (0.5 μg/rat/day, s.c.) or VD3*

17β-E2, fluoxetine hydrochloride and VD3 as cholecalciferol were provided from Sigma Chemical Co. (St. Louis, MO, USA). The solution of female estrogen was prepared using sterile sesame oil. Vitamin D3 was dissolved in 95% ethanol and then aliquoted and remained at −80°C. The solution of cholecalciferol for the injection into the experimental groups was diluted in sterile water, resulting in a solvent of VD3 containing 2% ethanol. Fluoxetine hydrochloride was dissolved in sterile physiological saline. All drugs were injected subcutaneously (0.1 ml/rat) for the 4 weeks during the CUMS procedure – 30 min before the daily stressor action – and throughout the period of the behavioral tests. All behavioral measurements were

All animals were randomly assigned to the six experimental groups (n = 7 in each): non-CUMS SHAM rats treated with solvent (control), SHAM rats exposed to CUMS treated with solvent, long-term OVX rats exposed to CUMS given with solvent, fluoxetine as positive control (10.0 mg/kg/day), 17β-E2 (0.5 μg/rat/day, s.c.) or VD3 (5.0 mg/ kg/day, s.c.) in a combination with low dose of 17β-E2. In our preliminary studies, there were no significant differences between SHAM/OVX rats treated with physiological

The total scheme of whole experiment is indicated on **Figure 1**.

made 60 min after the last drug administration.

*(5.0 mg/kg/day, s.c.) in a combination with low dose of 17β-E2.*

**156**

**2.4 Drugs**

**Figure 1.**

**2.5 Groups of animals**

We performed SPT accordingly to our previous study [50, 51]. Before and after the initiation of the 4 weeks CUMS procedures, the experimental rats were subjected to the sucrose preference test (SPT) [42, 51]. This test is set up as follows: following a training trial, the rats are subjected to a 24 h deprivation of food and water. On the next day, the rats have 1 hour access to one bottle with 200 ml of water and a similar amount of sucrose solution. The experimenter measures the percentage of the consumed sucrose solution and water volumes as a measure of sucrose preference by calculating the value of the sucrose preference among all (sucrose plus water in mL) liquid consumption:

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#### **2.7 Forced swimming test**

For testing of modifications of depression-like behavior, all groups of rats were submitted to the standard forced swimming test (FST) as described in earlier works (FST) [42, 43]. The three cylinders (60 cm tall and diameter 20 cm) were filled with 23–25°C water up to a 30-cm depth. On the first day, rats were pre-tested during 15 min in cylinders. Then, rats were dried with papers and placed at their home cages till the next day. On the second day (testing trial), OVX females with CUMS were examined into the apparatus for 5 min. The following parameters were registered: (1) immobility time (floating in the water with only movements necessary to keep the head above water); (2) swimming time (active swimming movements around glass cylinder); (3) climbing time (active movements with forepaws directed toward the walls). For recording of these values, a video camera was installed above the apparatus.

#### **2.8 Open field test**

The measurements of the behavioral activity in the OFT were carried out in a similar way to the method which has been published in a previous study [43]. The rats were set in the center square of the OFT and tested for 5 min. Motor activity and rearing and grooming behavior were recorded for 300 s in the OFT apparatus using a video camera, and equipment was cleaned in-between sessions.

#### **2.9 Biochemical measurements**

All rats underwent a narcosis after behavioral trials, and approximately 5 ml samples of blood were drawn from the animals to be centrifuged at 4000 *g* for

15 min at 4°C [43]. While doing so, the hippocampi of rats in the experimental group were dissected to be homogenized in cold lysis extraction buffer (0.2% sodium deoxycholate, 0.5% Triton X-100, 1% NP-40, 50 mM Tris-HCl pH 7.4, 1 mM phenylmethylsulfonyl fluoride, 1 mM N-ethyl-maleimide, and 2.5 mM phenanthroline) [43]. After that, the hippocampal samples with the cold lysis buffer were sonicated for 15 s. Then, the hippocampi were centrifuged at 12,000 *g* for 15 min at 4°C. The Bradford method was used for the normalization of hippocampal supernatants to the total protein [52]. The serum samples and hippocampal protein normalized supernatants were stored at −80°C until the ELISA assays. The serum samples were used for the measurement of the 25-hydroxyvitamin D3 (25- OH-VD3) and estradiol levels using a commercially available rat ELISA kits (Cusabio Biotech Co., Ltd., Wuhan, P.R. China) according to the manufacturer's instructions. The sensitivity and detection range of the 25-OH-VD3 rat ELISA kits were 5.0 μg/l and 20–100 μg/l, respectively. The sensitivity and detection range of the estradiol rat ELISA kits were 4.0 pg/ml and 40–1500 pg/ml, respectively.

Hippocampal homogenates were used for the detection of the NF-kB/p65/p65, 17β-E2/VD3 receptors levels by rat ELISA kits (Cusabio Biotech Co., Ltd., Wuhan, P.R. China) according to the manufacturer's instructions. Briefly, 100 l of hippocampal sample or standard was added to each well and incubated for 120 min at 37.0°C. Then, 100 l of anti-NF-kB/p65/p65, anti-17β-E2 receptor, and anti-VD3 receptor antibodies were added to each different well and incubated for 60 min at 37.0°C. After 3 times of washing, 100 l of HRP-avidin working solution was added to each well and incubated for 60 min at 37.0°C. Again, after 5 times of washing, 90 l of tetramethylbenzidine solution was given to each different well and incubated for 15–30 min at 37.0°C. Then, 50 l of stop solution was added to each well to terminate the color reaction. The NF-kB/p65/p65, 17β-E2/VD3 receptors levels were measured using a MC Thermo Fisher Scientific reader (Thermo Fisher Scientific Inc., Finland) with an absorbance of 450 nm. The standard curve was used for the calculation of the relationship between the optical density and the NF-kB/p65/p65, 17β-E2/VD3 receptors levels. The BDNF content is presented as pg/mg of tissue. The sensitivity and detection range of the NF-kB/p65 rat ELISA kit were 5.0 μg/ ml and 6.0–600 μg/ml, respectively. The sensitivity and detection range of the 17β-E2 receptor rat ELISA kit was 0.39 pg/ml and 1.56–100 pg/ml, respectively. The sensitivity and detection range of the VD3 receptor rat ELISA kit was 5.8 pg/ml and 23.5–1500 pg/ml, respectively. The assay exhibited no significant cross reactivity with other ligands. All samples were duplicated for the assay.

### **2.10 Western blots**

Hippocampal tissues were homogenized in cold lysis buffer containing a protease inhibitor cocktail (Sigma-Aldrich, USA) for 1 h and centrifuged at 12,000 *g* at 4°C for 20 min [42]. The protein content was evaluated by a Bio-Rad protein detector (Bio-Rad, USA), and 100 *μ*g of total protein from each sample was denatured with buffer (6.205 mM Tris-HCl, 10% glycerol, 2% SDS, 0.01% bromophenol blue, and 50 mM 2ME) at 95°C for 5 min. The denatured proteins were separated on an SDS page (10% sodium dodecyl sulfate polyacrylamide gel) and forwarded to a nitrocellulose membrane (Amersham Biotech, USA). After that, the membranes were probed with anti-NF-kB/p65/p65, anti-17β-E2 receptor, anti-VD3 receptor (1:1000, Santa Cruz), and β-actin (1:1000; Sigma-Aldrich, USA) monoclonal antibodies for 2 h and secondary antirabbit antibodies (1:5000; Santa Cruz, USA) conjugated to horseradish peroxidase for 1 h. Bands were detected by 5-bromo-4-chloro-3-indolyl phosphate with a nitro blue tetrazolium kit (Abcam, China) as a chemiluminescent substrate. Signals were measured by an image analysis system (UVIdoc, Houston, TX, USA).

**159**

**Figure 2.**

*Vitamin D3 Modulates NF-kB/p65, 17β-Estradiol, and Vitamin D Receptors Expression…*

USA). A value of P < 0.05 was considered statistically significant.

All experimental data are expressed as the mean ± standard deviation of the mean. The treatment effects were determined with a one-way ANOVA followed by an LSD *post hoc* test using the Statistics Package for SPSS, version 16.0 (SPSS Inc.,

**3.1 VD3 alters the body weight in the long-term OVX rats treated with 17β-E2**

The body weights of long-term OVX rats subjected to CUMS and treated with 17β-E2 in a combination with all investigated doses of VD3 are presented in **Figure 2**. There was no difference in the initial body weight in all the experimental groups. Following 4 weeks, the body weight of SHAM rats with CUMS was significantly decreased compared to the control, non-CUMS SHAM group (**Figure 2**, F(1,34) = 72.66, P < 0.001). The body weight of long-term OVX rats with CUMS was significantly decreased compared to the non-CUMS/ CUMS SHAM groups (**Figure 2**, P < 0.001). Administration of 17β-E2 did not statistically enhance body weight of long-term OVX rats with CUMS compared to the non-CUMS control, CUMS OVX/SHAM groups (**Figure 2**, P > 0.001). However, there was a tendency to increase the body weight of long-term OVX

*VD3 corrects the body weight in the long-term OVX rats treated with 17β-E2 submitted to CUMS: (a) Prior to CUMS and (b) After CUMS. \* – P < 0.05 versus the control group, # – P < 0.05 versus to the SHAM group with CUMS, \$ – P < 0.05 versus to the OVX group with CUMS, and \$\$ – P < 0.05 versus to the OVX group* 

*with CUMS treated with 17β-E2. The data are presented as mean ± SD; n = 7 in each group.*

*DOI: http://dx.doi.org/10.5772/intechopen.89357*

**2.11 Statistical analysis**

**exposed to CUMS**

**3. Results**

*Vitamin D3 Modulates NF-kB/p65, 17β-Estradiol, and Vitamin D Receptors Expression… DOI: http://dx.doi.org/10.5772/intechopen.89357*

#### **2.11 Statistical analysis**

All experimental data are expressed as the mean ± standard deviation of the mean. The treatment effects were determined with a one-way ANOVA followed by an LSD *post hoc* test using the Statistics Package for SPSS, version 16.0 (SPSS Inc., USA). A value of P < 0.05 was considered statistically significant.
