**4. Discussion**

The present preclinical study analyzed the antidepressant-like effects of VD3 (5.0 mg/kg, s.c.) in long-term adult OVX female rats given with low dose of 17β-E2 subjected to the CUMS. A CUMS paradigm is a well-known experimental paradigm that has documented to consider as standard pathophysiological impairments in mood state linked to a clinical depressive disorders in humans [47–49]. In the present work, the implications of NF-kB/p65 signaling pathway, as well as the 17β-E2/ VD3 receptors, in the mechanisms of VD3 activity in depression were tested regarding to the affective-related condition of long-term adult OVX rats treated with low dose of 17β-E2 exposed to CUMS.

The results of this study showed that in the adult long-term OVX rats undergoing CUMS, there were marked anhedonia-/depression-like behaviors, as assessed by SPT and LDT, respectively.

Moreover, long-term OVX rats exposed to CUMS exhibited decreased locomotor and rearing activities in the OFT. The ELISA assay clearly demonstrated lower estradiol and VD3 concentrations in adult long-term OVX rats subjected to CUMS. In addition, the increased NF-kB/p65 concentration/protein expression and decreased 17β-E2/VD3 receptors levels were found in the hippocampus of long-term OVX rats exposed to CUMS.

Administration of 17β-E2 failed to completely restore behavioral and biochemical parameters in the long-term OVX rats exposed to CUMS. Fluoxetine decreased anhedonia-like and depression-like states and decreased NF-kB/p65 levels in the hippocampus of the long-term OVX female rats exposed to CUMS. Data of literature have demonstrated that fluoxetine corrected depression-like profile of OVX rats in stress depression model [53]. The results of the study indicate that CUMS provokes marked behavioral, neurochemical, neurohormonal, and neuroinflammation alterations in adult OVX rats with long-lasting estrogens decline. Our data are in agreement with our recent data and other findings, which indicated that longterm estrogen deprivation in female rodents subjected to a CUMS procedure results in a profound affective-like profile [54].

The most important findings of the present study is linked to the antidepressantlike effects of VD3 in the long-term adult OVX rats treated with low dose of 17β-E2 under conditions of CUMS. VD3 given with a dose of 5.0 mg/kg reversed anhedonia-like and depression-like states in the SPT/LDT paradigms in the long-term OVX rats treated with 17β-E2 subjected to CUMS, which was similar to the effects of the fluoxetine treatment. Moreover, the VD3 application reversed the behavioral impairments observed in the OFT in the long-term OVX rats supplemented with 17β-E2 subjected to CUMS. Biochemical assays found that VD3 increased the serum VD3 and estradiol levels, as well decreased the hippocampal NF-kB/p65 content in the long-term OVX rats treated with 17β-E2 exposed to CUMS. Additionally, VD3

**167**

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

increased 17β-E2/VD3 receptors levels in the hippocampus of long-term OVX rats treated with 17β-E2 subjected to CUMS. Western blot analysis revealed that VD3 reduced NF-kB/p65 and increased 17β-E2/VD3 protein expression in the hippocampus of long-term OVX rats treated with 17β-E2 subjected to CUMS. These data suggest that VD3 attenuates the CUMS-produced behavioral impairments and normalized the serum VD3 and estradiol levels, as well NF-kB/p65 and 17β-E2/VD3

Inflammation is now recognized to be the one of the key components of affective-related development, with the NF-κB involved in both the early and late stages of the inflammatory processing [33, 41, 55]. NF-κB is the main transcriptional factor which controls the expression of various genes implicated in multiple cell functions and is triggered by different types of extracellular stimuli stimulated neuroinflammation [33, 34]. Deterioration of NF-κB signaling in the brain negatively influence on neuroplasticity and neuromorphology, as well as cognitive functions. However, NF-κB overstimulation is deleterious, and this detrimental effect can be reversed suppressing NF-κB signaling. That is why, NF-κB signaling is fundamental for normal brain function [36–39, 55]. The inhibition of neuroinflammation may be critical for the antidepressant action of VD3 that was noted in our study. The increased pro-inflammatory cytokines have been found repeatedly in both animals and depressed patients [37–39]. Clinical studies indicate that inhibition of neuroinflammation by nonsteroidal antiinflammatory drugs can attenuate depression-like behaviors in depressed rodents and humans [36–39]. The inhibitory effect of VD3 on CUMS-induced increase in pro-inflammatory cytokines in the hippocampus of the long-term OVX rats is strongly in accordance with the neuroinflammation hypothesis of depression [39]. A better comprehension of NFκB-dependent mecha-

Based on our findings, it can be assumed that VD3 in the present study is implicated in the modulation of NF-κB signaling in long-term OVX rats with CUMS. On the other hand, VD3 normalized 17β-E2/VD3 receptors levels in the hippocampus of long-term OVX rats treated with 17β-E2 subjected to CUMS. Such complex effects of VD3 on neuroinflammation and 17β-E2/VD3 receptors might promote a greater effect of combination of VD3 plus 17β-E2 than application only 17β-E2. This is the first study to show the action of VD3 in the behavioral and neuroinflammation and biochemical consequences of a CUMS in adult long-term OVX rats treated with low dose of 17β-E2. The inhibition of NF-kB/p65 activity by VD3 treatment is a promising fact of study for treatment of neuroinflammatory diseases that are associated with low levels of VD3. These results suggest an anti-inflammatory role for VD3,

Thus, the possible mechanism of VD3 action might be explained by the stimulation of 17β-E2/VD3 receptors identified in the different brain structures involved in mood control [13–15]. The possible mechanisms of such action of VD3 in the long-term OVX rats can be connected with cross-talk protein-protein interactions. Moreover, VD alters the neuroinflammation response via NF-kB/p65 signaling at the affective-related state, thereby improving depression state [39, 55]. Low VD levels appear in the majority of postmenopausal women [52, 56, 57]. Therefore, VD supplementation may be very useful for treatment of mood disorders in postmenopausal women with a low level of VD and supplemented with MHT. However, the exact role of VD supplementation in the prevention and treatment of mood disorders associated with menopausal consequences has not been completely identified. In conclusion, the present study supports evidence for repeated administration of VD3 in a chronic unpredictable stress model having an anti-anhedonia-like and antidepressant-like effects in long-term OVX adult rats treated with low dose of 17β-E2. Moreover, the biochemical and western blotting assays suggest the

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

production in the hippocampus of long-term OVX rats.

nisms in antidepressant action of VD3 needs further studies.

which may be one of the fundamental components of its activity.

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

increased 17β-E2/VD3 receptors levels in the hippocampus of long-term OVX rats treated with 17β-E2 subjected to CUMS. Western blot analysis revealed that VD3 reduced NF-kB/p65 and increased 17β-E2/VD3 protein expression in the hippocampus of long-term OVX rats treated with 17β-E2 subjected to CUMS. These data suggest that VD3 attenuates the CUMS-produced behavioral impairments and normalized the serum VD3 and estradiol levels, as well NF-kB/p65 and 17β-E2/VD3 production in the hippocampus of long-term OVX rats.

Inflammation is now recognized to be the one of the key components of affective-related development, with the NF-κB involved in both the early and late stages of the inflammatory processing [33, 41, 55]. NF-κB is the main transcriptional factor which controls the expression of various genes implicated in multiple cell functions and is triggered by different types of extracellular stimuli stimulated neuroinflammation [33, 34]. Deterioration of NF-κB signaling in the brain negatively influence on neuroplasticity and neuromorphology, as well as cognitive functions. However, NF-κB overstimulation is deleterious, and this detrimental effect can be reversed suppressing NF-κB signaling. That is why, NF-κB signaling is fundamental for normal brain function [36–39, 55]. The inhibition of neuroinflammation may be critical for the antidepressant action of VD3 that was noted in our study. The increased pro-inflammatory cytokines have been found repeatedly in both animals and depressed patients [37–39]. Clinical studies indicate that inhibition of neuroinflammation by nonsteroidal antiinflammatory drugs can attenuate depression-like behaviors in depressed rodents and humans [36–39]. The inhibitory effect of VD3 on CUMS-induced increase in pro-inflammatory cytokines in the hippocampus of the long-term OVX rats is strongly in accordance with the neuroinflammation hypothesis of depression [39]. A better comprehension of NFκB-dependent mechanisms in antidepressant action of VD3 needs further studies.

Based on our findings, it can be assumed that VD3 in the present study is implicated in the modulation of NF-κB signaling in long-term OVX rats with CUMS. On the other hand, VD3 normalized 17β-E2/VD3 receptors levels in the hippocampus of long-term OVX rats treated with 17β-E2 subjected to CUMS. Such complex effects of VD3 on neuroinflammation and 17β-E2/VD3 receptors might promote a greater effect of combination of VD3 plus 17β-E2 than application only 17β-E2. This is the first study to show the action of VD3 in the behavioral and neuroinflammation and biochemical consequences of a CUMS in adult long-term OVX rats treated with low dose of 17β-E2. The inhibition of NF-kB/p65 activity by VD3 treatment is a promising fact of study for treatment of neuroinflammatory diseases that are associated with low levels of VD3. These results suggest an anti-inflammatory role for VD3, which may be one of the fundamental components of its activity.

Thus, the possible mechanism of VD3 action might be explained by the stimulation of 17β-E2/VD3 receptors identified in the different brain structures involved in mood control [13–15]. The possible mechanisms of such action of VD3 in the long-term OVX rats can be connected with cross-talk protein-protein interactions. Moreover, VD alters the neuroinflammation response via NF-kB/p65 signaling at the affective-related state, thereby improving depression state [39, 55]. Low VD levels appear in the majority of postmenopausal women [52, 56, 57]. Therefore, VD supplementation may be very useful for treatment of mood disorders in postmenopausal women with a low level of VD and supplemented with MHT. However, the exact role of VD supplementation in the prevention and treatment of mood disorders associated with menopausal consequences has not been completely identified.

In conclusion, the present study supports evidence for repeated administration of VD3 in a chronic unpredictable stress model having an anti-anhedonia-like and antidepressant-like effects in long-term OVX adult rats treated with low dose of 17β-E2. Moreover, the biochemical and western blotting assays suggest the

*Vitamin D Deficiency*

**4. Discussion**

dose of 17β-E2 exposed to CUMS.

SPT and LDT, respectively.

OVX rats exposed to CUMS.

in a profound affective-like profile [54].

females (**Figure 8**, P < 0.05). NF-kB/p65 levels were increased in the hippocampus of long-term OVX rats with CUMS compared to the non-CUMS/CUMS SHAM rats (**Figure 8**, F(1,34) = 34.45, F(1,34) = 16.38, respectively, P < 0.05). Fluoxetine (10.0 mg/kg) resulted in significant reduced levels of hippocampal NF-kB/p65 protein expression in long-term OVX with CUMS compared to the OVX plus solvent/SHAM rats with CUMS (**Figure 8**, P < 0.05). Co-treatment with VD3 and 17β-E2 decreased NF-kB/p65 protein levels and increased 17β-E2/VD3 receptors protein expression in the hippocampus of the long-term OVX rats compared to OVX plus solvent or 17β-E2/ SHAM rats with CUMS (**Figure 8**, P < 0.05). Fluoxetine did not alter 17β-E2/VD3 protein expression in the long-term OVX rats exposed to CUMS (**Figure 8**, P > 0.05).

The present preclinical study analyzed the antidepressant-like effects of VD3 (5.0 mg/kg, s.c.) in long-term adult OVX female rats given with low dose of 17β-E2 subjected to the CUMS. A CUMS paradigm is a well-known experimental paradigm that has documented to consider as standard pathophysiological impairments in mood state linked to a clinical depressive disorders in humans [47–49]. In the present work, the implications of NF-kB/p65 signaling pathway, as well as the 17β-E2/ VD3 receptors, in the mechanisms of VD3 activity in depression were tested regarding to the affective-related condition of long-term adult OVX rats treated with low

The results of this study showed that in the adult long-term OVX rats undergoing CUMS, there were marked anhedonia-/depression-like behaviors, as assessed by

Administration of 17β-E2 failed to completely restore behavioral and biochemical parameters in the long-term OVX rats exposed to CUMS. Fluoxetine decreased anhedonia-like and depression-like states and decreased NF-kB/p65 levels in the hippocampus of the long-term OVX female rats exposed to CUMS. Data of literature have demonstrated that fluoxetine corrected depression-like profile of OVX rats in stress depression model [53]. The results of the study indicate that CUMS provokes marked behavioral, neurochemical, neurohormonal, and neuroinflammation alterations in adult OVX rats with long-lasting estrogens decline. Our data are in agreement with our recent data and other findings, which indicated that longterm estrogen deprivation in female rodents subjected to a CUMS procedure results

The most important findings of the present study is linked to the antidepressantlike effects of VD3 in the long-term adult OVX rats treated with low dose of 17β-E2 under conditions of CUMS. VD3 given with a dose of 5.0 mg/kg reversed anhedonia-like and depression-like states in the SPT/LDT paradigms in the long-term OVX rats treated with 17β-E2 subjected to CUMS, which was similar to the effects of the fluoxetine treatment. Moreover, the VD3 application reversed the behavioral impairments observed in the OFT in the long-term OVX rats supplemented with 17β-E2 subjected to CUMS. Biochemical assays found that VD3 increased the serum VD3 and estradiol levels, as well decreased the hippocampal NF-kB/p65 content in the long-term OVX rats treated with 17β-E2 exposed to CUMS. Additionally, VD3

Moreover, long-term OVX rats exposed to CUMS exhibited decreased locomotor and rearing activities in the OFT. The ELISA assay clearly demonstrated lower estradiol and VD3 concentrations in adult long-term OVX rats subjected to CUMS. In addition, the increased NF-kB/p65 concentration/protein expression and decreased 17β-E2/VD3 receptors levels were found in the hippocampus of long-term

**166**

implications of NF-kB/p65 and 17β-E2/VD3 production modulation in the antidepressant-like activity of VD3. Further studies should however explore the precise mechanism of VD3 action, due to the necessity of an improvement of therapies focusing on mood-repair in females with long-lasting estrogen deficiency.
