**3. Results**

*Vitamin D Deficiency*

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.

with other ligands. All samples were duplicated for the assay.

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

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).

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**2.10 Western blots**

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

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

#### **Figure 2.**

*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.*

rats with CUMS compared to the OVX rats plus CUMS given with solvent. Supplementation with VD3 (5.0 mg/kg) plus 17β-E2 significantly prevented the reduction of the body weight of long-term OVX rats with CUMS (P < 0.001) compared to the OVX plus solvent or 17β-E2/SHAM rats exposed to CUMS (**Figure 2**, P < 0.001). This effect of co-administration of VD3 (5.0 mg/kg) plus 17β-E2 was similar to the effect of the reference drug fluoxetine (10.0 mg/kg) in long-term OVX rats with CUMS.
