J Endocrinol Invest (2014) 37:1109–1116 DOI 10.1007/s40618-014-0191-5
ORIGINAL ARTICLE
Chronic stress causes neuroendocrine-immune disturbances without affecting renal vitamin D metabolism in rats P. Jiang · L. Zhang · W. Zhu · H. Li · R. Dang · M. Tang
Received: 22 July 2014 / Accepted: 6 October 2014 / Published online: 16 October 2014 © Italian Society of Endocrinology (SIE) 2014
Abstract Introduction Vitamin D (VD) insufficiency has been repeatedly observed in the medical conditions associated with inflammation, such as cardiovascular disease, diabetes and depression. However, contrasting to the observational evidence, randomized trials of VD supplementation failed to demonstrate such link. Given the recent evidence that the inflammatory process can in turn alter VD metabolism, it has been hypothesized that the insufficient VD status could be the result rather than the cause of chronic inflammation involved in the onset of depression and other disease conditions. Materials and methods Chronic mild stress (CMS) is a valid animal model of depression that accompanied with neuroendocrine-immune disturbances. In the present research, we assessed serum VD concentrations and renal expression of the cytochromes P450 enzymes involved in VD activation (CYP27B1) and catabolism (CYP24A1) of rats following 8-week exposure to CMS. Results While CMS induced the rats to a depression-like state and increased serum levels of the proinflammatory cytokines and corticosterone, and the antidepressant, sertraline, mitigated depression-like behaviors and neuroendocrine-immune disturbances, neither the stress regimen nor sertraline significantly affected endocrine metabolism of VD. Conclusion Our data suggest that the stress-induced neuroendocrine-immune disturbances may account for the development of depression, but are not responsible for the
P. Jiang · L. Zhang · W. Zhu · H. Li (*) · R. Dang · M. Tang Institute of Clinical Pharmacy and Pharmacology, Second Xiangya Hospital, Central South University, Changsha, China e-mail: [email protected]
insufficient VD status that frequently observed in depressed patients. Keywords Stress · Depression · Cytokines · Corticosterone · Vitamin D metabolism
Introduction Vitamin D (VD) now is recognized as a secosteroid hormone affecting multiple aspects of human physiology, and the actions of VD have extended far beyond its classical role in calcium-phosphate homeostasis and bone metabolism. The poor VD status has been repeatedly observed in a variety of chronic illnesses, such as cardiovascular disease, diabetes mellitus, inflammatory bowel disease and neuropsychiatric disorders [1, 2]. Interestingly, inflammation actively participates in these medical conditions, whereas VD is known to have several immunomodulatory functions, including suppression of proinflammatory cytokines and regulation of immune cell activity [3, 4]. The role of VD in maintaining immune homeostasis has become a cornerstone of the underlying association between VD deficiency and increased risk for development of the inflammatory related diseases. However, in clinical studies, the causal relationship between VD and the multiple health outcomes does not seem to be that simple. Although observational studies have strongly linked VD to a variety of diseases, the randomized controlled trial, the golden standard design to establish a causal relationship and an outcome, mostly failed to obtain a positive outcome from VD supplementation [5, 6]. The contrasting results from epidemiological data and intervention studies lead to the hypothesis that VD deficiency might not be the cause but an accompanied state.
13
1110
VD is either ingested in the diet or generated de novo through scission of 7-hydrocholesterol in the skin by ultraviolet light. In hepatic cells, VD is hydroxylated into its major circulating metabolite, 25-hydroxyvitamin D (25(OH) D). Mainly in the kidney, 1α-hydroxylase (CYP27B1) turns the circulating 25(OH)D into its active form, 1,25-dihydroxyvitamin D (1,25(OH)2D), which is then catabolized by 24-hydroxylase (CYP24A1) [7]. Many factors, such as season, latitude, physical activity, aging, adiposity and diet can affect circulating VD status [8]. Thus, low concentrations of VD could be attributed to many of these cofounding effects. However, prospective studies provide evidence that is less likely to be contaminated by reverse causality, and after controlling for a variety of confounding factors, several longitudinal prospective cohort studies still support the notion that hypovitaminosis D may contribute causally to many chronic diseases, including depression [9, 10]. In this scenario, the key question still remains whether hypovitaminosis D is the cause or simply a marker of some other processes that are responsible for the disease progression. It should be noted that almost all of these chronic non-skeletal disorders that associated with VD share one common pathophysiologic mechanism, i.e. inflammation. Although well-documented data have pointed out the immunomodulatory properties of VD, recent studies also raised the possibility that chronic inflammation may in turn alter VD endocrine metabolism [11]. Depression is associated with a chronic inflammation, and increased levels of proinflammatory cytokines—such as IL-1β, TNF-α, IL-6 and IFN-γ—have been repeatedly observed in depressed individuals [12]. An even stronger association between the inflammation and the depressive behaviors has been reported in rodent studies where administration of IL-1β, TNF-α and IL-6 resulted in depressionlike behaviors [13]. Additionally, genetic variations of IL-1β, TNF-α, IL-6 and IFN-γ are associated with the susceptibility to depression and the responsiveness to antidepressive medication [14]. Psychological, as well as physical stress can activate immune response and inflammation process, and lead to increased cytokine levels, contributing to structural and functional alterations of neurons and the development of depression [13]. Notably, depressive patients are prone to have low VD status and are at a risk of bone loss. While there is evidence that VD may have anti-stress activity [15], the evidence considering whether the stress-induced neuroendocrine-immune disturbances can in turn alter VD endocrine actions remains equivocal. Thus, in the present study, we assessed serum VD concentrations and renal expression of the cytochromes P450 enzymes involved in VD activation (CYP27B1) and catabolism (CYP24A1) in response to chronic mild stress (CMS). The effect of sertraline, a widely used selective serotonin reuptake inhibitor (SSRI) antidepressant drug, on the CMS-induced activation of inflammation and endocrine metabolism of VD was also evaluated.
13
J Endocrinol Invest (2014) 37:1109–1116
Materials and methods Animals Male, Sprague–Dawley rats (200–230 g), supplied by the Experimental Animal Center of the Second Xiangya Hospital, were housed under standard conditions of temperature (23 ± 2 °C) and light (12:12 h light/dark cycle), with free access to food and water, except prior to sucrose preference test (SPT) or when they were submitted to chronic mild stress (CMS) (see below). All animal use procedures were carried out in accordance with the Regulations of Experimental Animal Administration issued by the State Committee of Science and Technology of the People’s Republic of China, with the approval of the Ethics Committee in our university. Chronic stress paradigm and drug treatment Rats were randomly allocated to one of the four groups (n = 10): vehicle (control), control + sertraline, CMS and CMS + sertraline. The animals in different groups received daily gavage of 8 mg/kg sertraline or same volume of saline or (and) one of the following stressors for 8 weeks: soiled cages for 24 h, cage tilt (45°) for 24 h, immobilization for 2 h, tail clamping for 2 min, day–night reversal (12 h/12 h), food deprivation for 24 h and exposure to an empty water bottle for 1 h immediately following 24 h of water deprivation. Sucrose preference test (SPT) SPT is a measure of stressed-induced anhedonia state, a key depressive-like behavior in rats [16]. Prior to SPT, all the rats were housed individually and habituated to 48 h of forced 1 % sucrose solution consumption in two bottles on each side. Then after 16 h water deprivation, we placed two pre-weighted bottles, one containing 1 % sucrose solution and another containing tap water to each rat. The side (left and right) of the two bottles was randomly placed, to avoid spatial bias. The bottles were weighted again after 1 h and the weight difference was considered to be the rat intake from each bottle. The preference for sucrose was measured as a percentage of the consumed 1 % sucrose solution relative to the total amount of liquid intake. Tissue collection and serum biochemical assays One day after the behavioral test, rats were anesthetized with 10 % chloral hydrate (4 ml/kg). Trunk blood was collected and the tissues were quickly removed, chilled in liquid nitrogen and then stored at −80 °C. The left side of the kidney was used for PCR test, whereas the right kidney was
J Endocrinol Invest (2014) 37:1109–1116
1111
used for western blotting analysis. Commercially available Enzyme-Linked Immunosorbent Assay (ELISA) kits were used to measure serum proinflammatory cytokines: IL-1β (R&D Systems, USA), IL-6 (Cusabio, China), TNFα (Cusabio, China) and IFNγ (R&D Systems, USA). Serum status of corticosterone and VD were also quantified by ELISA method: corticosterone (Cayman Chemical, USA), 25(OH) D (Cusabio, China) and 1,25(OH)2D (Immunodiagnostic Systems, UK).
cytokine and hormone status were determined by one-way ANOVA test followed by LSD test for post hoc comparisons. The prior level of significance was established at P
ORIGINAL ARTICLE
Chronic stress causes neuroendocrine-immune disturbances without affecting renal vitamin D metabolism in rats P. Jiang · L. Zhang · W. Zhu · H. Li · R. Dang · M. Tang
Received: 22 July 2014 / Accepted: 6 October 2014 / Published online: 16 October 2014 © Italian Society of Endocrinology (SIE) 2014
Abstract Introduction Vitamin D (VD) insufficiency has been repeatedly observed in the medical conditions associated with inflammation, such as cardiovascular disease, diabetes and depression. However, contrasting to the observational evidence, randomized trials of VD supplementation failed to demonstrate such link. Given the recent evidence that the inflammatory process can in turn alter VD metabolism, it has been hypothesized that the insufficient VD status could be the result rather than the cause of chronic inflammation involved in the onset of depression and other disease conditions. Materials and methods Chronic mild stress (CMS) is a valid animal model of depression that accompanied with neuroendocrine-immune disturbances. In the present research, we assessed serum VD concentrations and renal expression of the cytochromes P450 enzymes involved in VD activation (CYP27B1) and catabolism (CYP24A1) of rats following 8-week exposure to CMS. Results While CMS induced the rats to a depression-like state and increased serum levels of the proinflammatory cytokines and corticosterone, and the antidepressant, sertraline, mitigated depression-like behaviors and neuroendocrine-immune disturbances, neither the stress regimen nor sertraline significantly affected endocrine metabolism of VD. Conclusion Our data suggest that the stress-induced neuroendocrine-immune disturbances may account for the development of depression, but are not responsible for the
P. Jiang · L. Zhang · W. Zhu · H. Li (*) · R. Dang · M. Tang Institute of Clinical Pharmacy and Pharmacology, Second Xiangya Hospital, Central South University, Changsha, China e-mail: [email protected]
insufficient VD status that frequently observed in depressed patients. Keywords Stress · Depression · Cytokines · Corticosterone · Vitamin D metabolism
Introduction Vitamin D (VD) now is recognized as a secosteroid hormone affecting multiple aspects of human physiology, and the actions of VD have extended far beyond its classical role in calcium-phosphate homeostasis and bone metabolism. The poor VD status has been repeatedly observed in a variety of chronic illnesses, such as cardiovascular disease, diabetes mellitus, inflammatory bowel disease and neuropsychiatric disorders [1, 2]. Interestingly, inflammation actively participates in these medical conditions, whereas VD is known to have several immunomodulatory functions, including suppression of proinflammatory cytokines and regulation of immune cell activity [3, 4]. The role of VD in maintaining immune homeostasis has become a cornerstone of the underlying association between VD deficiency and increased risk for development of the inflammatory related diseases. However, in clinical studies, the causal relationship between VD and the multiple health outcomes does not seem to be that simple. Although observational studies have strongly linked VD to a variety of diseases, the randomized controlled trial, the golden standard design to establish a causal relationship and an outcome, mostly failed to obtain a positive outcome from VD supplementation [5, 6]. The contrasting results from epidemiological data and intervention studies lead to the hypothesis that VD deficiency might not be the cause but an accompanied state.
13
1110
VD is either ingested in the diet or generated de novo through scission of 7-hydrocholesterol in the skin by ultraviolet light. In hepatic cells, VD is hydroxylated into its major circulating metabolite, 25-hydroxyvitamin D (25(OH) D). Mainly in the kidney, 1α-hydroxylase (CYP27B1) turns the circulating 25(OH)D into its active form, 1,25-dihydroxyvitamin D (1,25(OH)2D), which is then catabolized by 24-hydroxylase (CYP24A1) [7]. Many factors, such as season, latitude, physical activity, aging, adiposity and diet can affect circulating VD status [8]. Thus, low concentrations of VD could be attributed to many of these cofounding effects. However, prospective studies provide evidence that is less likely to be contaminated by reverse causality, and after controlling for a variety of confounding factors, several longitudinal prospective cohort studies still support the notion that hypovitaminosis D may contribute causally to many chronic diseases, including depression [9, 10]. In this scenario, the key question still remains whether hypovitaminosis D is the cause or simply a marker of some other processes that are responsible for the disease progression. It should be noted that almost all of these chronic non-skeletal disorders that associated with VD share one common pathophysiologic mechanism, i.e. inflammation. Although well-documented data have pointed out the immunomodulatory properties of VD, recent studies also raised the possibility that chronic inflammation may in turn alter VD endocrine metabolism [11]. Depression is associated with a chronic inflammation, and increased levels of proinflammatory cytokines—such as IL-1β, TNF-α, IL-6 and IFN-γ—have been repeatedly observed in depressed individuals [12]. An even stronger association between the inflammation and the depressive behaviors has been reported in rodent studies where administration of IL-1β, TNF-α and IL-6 resulted in depressionlike behaviors [13]. Additionally, genetic variations of IL-1β, TNF-α, IL-6 and IFN-γ are associated with the susceptibility to depression and the responsiveness to antidepressive medication [14]. Psychological, as well as physical stress can activate immune response and inflammation process, and lead to increased cytokine levels, contributing to structural and functional alterations of neurons and the development of depression [13]. Notably, depressive patients are prone to have low VD status and are at a risk of bone loss. While there is evidence that VD may have anti-stress activity [15], the evidence considering whether the stress-induced neuroendocrine-immune disturbances can in turn alter VD endocrine actions remains equivocal. Thus, in the present study, we assessed serum VD concentrations and renal expression of the cytochromes P450 enzymes involved in VD activation (CYP27B1) and catabolism (CYP24A1) in response to chronic mild stress (CMS). The effect of sertraline, a widely used selective serotonin reuptake inhibitor (SSRI) antidepressant drug, on the CMS-induced activation of inflammation and endocrine metabolism of VD was also evaluated.
13
J Endocrinol Invest (2014) 37:1109–1116
Materials and methods Animals Male, Sprague–Dawley rats (200–230 g), supplied by the Experimental Animal Center of the Second Xiangya Hospital, were housed under standard conditions of temperature (23 ± 2 °C) and light (12:12 h light/dark cycle), with free access to food and water, except prior to sucrose preference test (SPT) or when they were submitted to chronic mild stress (CMS) (see below). All animal use procedures were carried out in accordance with the Regulations of Experimental Animal Administration issued by the State Committee of Science and Technology of the People’s Republic of China, with the approval of the Ethics Committee in our university. Chronic stress paradigm and drug treatment Rats were randomly allocated to one of the four groups (n = 10): vehicle (control), control + sertraline, CMS and CMS + sertraline. The animals in different groups received daily gavage of 8 mg/kg sertraline or same volume of saline or (and) one of the following stressors for 8 weeks: soiled cages for 24 h, cage tilt (45°) for 24 h, immobilization for 2 h, tail clamping for 2 min, day–night reversal (12 h/12 h), food deprivation for 24 h and exposure to an empty water bottle for 1 h immediately following 24 h of water deprivation. Sucrose preference test (SPT) SPT is a measure of stressed-induced anhedonia state, a key depressive-like behavior in rats [16]. Prior to SPT, all the rats were housed individually and habituated to 48 h of forced 1 % sucrose solution consumption in two bottles on each side. Then after 16 h water deprivation, we placed two pre-weighted bottles, one containing 1 % sucrose solution and another containing tap water to each rat. The side (left and right) of the two bottles was randomly placed, to avoid spatial bias. The bottles were weighted again after 1 h and the weight difference was considered to be the rat intake from each bottle. The preference for sucrose was measured as a percentage of the consumed 1 % sucrose solution relative to the total amount of liquid intake. Tissue collection and serum biochemical assays One day after the behavioral test, rats were anesthetized with 10 % chloral hydrate (4 ml/kg). Trunk blood was collected and the tissues were quickly removed, chilled in liquid nitrogen and then stored at −80 °C. The left side of the kidney was used for PCR test, whereas the right kidney was
J Endocrinol Invest (2014) 37:1109–1116
1111
used for western blotting analysis. Commercially available Enzyme-Linked Immunosorbent Assay (ELISA) kits were used to measure serum proinflammatory cytokines: IL-1β (R&D Systems, USA), IL-6 (Cusabio, China), TNFα (Cusabio, China) and IFNγ (R&D Systems, USA). Serum status of corticosterone and VD were also quantified by ELISA method: corticosterone (Cayman Chemical, USA), 25(OH) D (Cusabio, China) and 1,25(OH)2D (Immunodiagnostic Systems, UK).
cytokine and hormone status were determined by one-way ANOVA test followed by LSD test for post hoc comparisons. The prior level of significance was established at P
