| Summary: | 碩士 === 國防醫學院 === 生理學研究所 === 89 === Abstract
Although 1,25-dihydroxyvitamin D3 (or Vit. D3), an active metabolite of vitamin D3, is well known for its systemic regulatory role in the maintenance of calcium homeostasis, recent evidence also indicates its neuroprotective and antioxidant potential in the central nervous system (CNS). The receptors for vitamin D (VDR) and Vit. D3 are also found in the CNS, suggesting central action site for both endogenous or exogenous Vit. D3.
Parkinson's disease (PD) is associated with oxidative stress and characterized by dopaminergic neuronal death in substantial nigra (SN) in the ventral mesencephalon. Insights into the pathogenesis of PD have been achieved experimentally by using the neurotoxin 6-hydroxy dopamine (6-OHDA). In this study we are interested in examining whether Vit. D3 has neuroprotective effect on 6-OHDA-induced toxicities in dopaminergic neurons. Based on reports showing activated glia cells in postmortem SN of PD patients, we are also interested in examining the effects of Vit. D3 on activation of glia cells. The immunoregulatory activities of glial cells (particularly microglia and astrocytes) are apparent following neuronal injuries. Among the potential mediators released during glial activation are nitric oxide (NO), reactive oxygen species (ROS) and the tumor necrosis factor a (TNFa), a proinflammatory cytokine and a signal of apoptosis and neural damage. It is known that ROS mediate the signal for NF-kB activation and results in production of NO (via induction of iNOS) and cytokines (such as TNFa) by the bacterial endotoxin lipopolysaccardie (LPS). Since ROS has been implicated in the pathogenesis of PD, 6-OHDA-induced toxicities of dopaminergic neurons, as well as activation of glial cells, Vit. D3 may protect dopaminergic neurons from these two aspects. In this thesis, dopaminergic neurons damaged by 6-OHDA and glial activation by LPS were used as models to elucidate the effects and mechanism of Vit. D3 in vitro. Primary cultures of ventromesencephalon (VM) and mixed glia were prepared from 14-15 day embryo and newborn of Sprague-Dawley rats, respectively. Following 8 days of incubation, VM cultures were pretreated with Vit. D3 for 48 hours and then treated with 6-OHDA (30 mM) for 1 hour. Mixed glial cultures were treated with LPS and Vit. D3 for 24 hours. ROS production and TNFa were measured after 24 hours. Cell identification and morphology was examined with immunocytochemical staining. Our results indicated that 6-OHDA toxicities were characterized by lactate dehydroxylase (LDH) activity in the culture medium, reduced length of neurite and survival number of TH (+) cells. Pretreatment with Vit. D3（5 x 10-11 or 10-10 M） attenuated dopaminergic neuronal loss and decreased neurite length induced by 6-OHDA. Pretreatment with Vit. D3（10-10 M）also attenuated the increase in ROS production, as measured by 2,7-dichlorofluorescin diacetate (DCF-DA), by 6-OHDA. Moreover, it has been reported that endogenous neurotrophic factors such as glial cell line-derived neurotrophic factor (GDNF) and nerve growth factor (NGF) can be released by Vit. D3. Consistent with the mitogen-activated protein kinase (MAPK) pathways is involved in the neurotrophic activities of growth factors possibly released by Vit. D3, the neuroprotective effect of Vit. D3 was antagonized by pretreatment with PD98059, a p42/44 MAPK inhibitor, but not SB202191 or SB203580, p38 MAPK inhibitors, at 10 μM. The neuroprotective effects of Vit. D3 against 6-OHDA toxicities was also attenuated by AG 879, a NGF receptor (TrkA) inhibitor, further supporting the notion that NGF is involved in mediating the neuroprotective effect of Vit. D3. The activation of glial cells was examined by measuring the production of NO, ROS and TNFa as functional indices. Following glial activation by LPS (100 ng/ml) in mixed glial culture, the increase in nitrite accumulation was attenuated by simultaneous treatment with Vit. D3 (10-7, 3x10-8 M) but not by pretreatment with Vit. D3 for 48 hours. LPS increased intercellular ROS production after 12 hours but decreased it at 24 hours. Vit. D3 decreased LPS induced ROS production and treatment with Vit. D3 alone for 3 hours has the potential to decrease intercellular ROS. LPS increased TNFa following treatment for 3 hours and decreased it after 12 hours. Vit. D3 (10-7, 3x10-8 M) decreased LPS- induced TNFa release from mixed glia.
In sum, our results indicated that Vit. D3 protected dopaminergic neuron against 6-OHDA toxicities in VM cultures. The mechanisms may involve, at least, the activation of the MAPK pathway by NGF and reduced production of ROS. Vit. D3 also suppressed the activation of glial cells as reflected by the decreased production of NO, TNFa and ROS. Taken together, Vit. D3 may have therapeutic potential for PD by its action on both dopaminergic neurons and glial cells. The decreased production of ROS may be involved in mediating the effects of Vit. D3 in both dopaminergic neurons and glial cells.
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