Estradiol And Raloxifene On Cultured Rat Hippocampal And Cortical Neuronal Injury In The Protective Effect

Estrogen has various physiological functions throughout the body, including in the central nervous system. It is well established that estrogen acts as a neuroprotector in neurodegenerative processes in vivo. In vitro studies also demonstrate that estrogen enhances neuronal survival in a variety of neuronal injuries including oxidative stress, excitotoxic insults and 6-amyloid toxicity. Moreover, epidemiological studies support a positive role for estrogen replacement therapy (ERT) in reducing the incidence of Alzheimer's disease (AD) in postmenopausal women. There is also evidence indicating that estrogen confers benefits against Parkinson's disease (PD). However, because of its association with an increasing risk of breast as well as uterine cancer, the use of ERT in postmenopausal women is still controversial. Therefore, there has been intensive interest in designing optimal estrogen-like molecules to be the safer alternatives than the traditional ERT.Raloxifene, as one of the selective estrogen receptor modulators (SERMs), is a synthetic non-steroidal benzothiophen derivative. It exerts as an estrogen receptor antagonist in the reproductive system to inhibit the growth of breast and uterine cancer, whereas in nonreproductive tissues, it acts as a partial estrogen receptor agonist to prevent bone loss and lower serum cholesterol. There is also evidence showing that raloxifene has estrogen agonistic activity on dopamine receptor in the central nervous system. In addition, raloxifene protects against oxidative stress and β-amyloid-induced neurotoxicity in vitro. The tissue-selective agonist and antagonist actions of raloxifene have led it possible to be an ideal drug in hormone replacement therapy. Although there is evidence indicating that raloxifene is neuropretective, the mechanism involved is incompletely understood.Glutamate is a principal excitatory amino acid neurotransmitter in the central nervous system. Activation of glutamate receptors, especially the N-methyl-D-aspartate (NMDA) receptor, also plays a pivotal role in the synaptic plasticity, which is thought to underlie learning and memory. Over-stimulation of the glutamate receptors, however, may trigger cytosolic Ca²⁺ increase, which will in turn results in neuronal apoptosis or necrosis. This is the glutamate-induced excitotoxic damage, which is important in many neurodegenerative process including stroke, AD, PD, Huntington's disease (HD), and hypoxia-ischemia brain injury. In addition to excitotoxic damage, many other damages caused by oxidative stress and β-amyloidtoxicity can be attributed to disturbance of Ca²⁺ homeostasis.To investigate whether 17β-estrsdiol and raloxifene affected the glutamate-induced calcium (Ca²⁺) overload in rat cultured neurons, experiments were carried out in rat primary cultured neurons. With the aid of confocal laser scanning microscope, the bulk intracellular Ca²⁺ level was measured in neurons with fluorescent Ca²⁺ probe fluo3. The cell viability was determined by 3-(4,5-dimthyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. And calcium currents were recorded with the use of whole cell patch-clamp.Part I Protection of 17β-estradiol against neuronal damage in rat cultured hippocampal neurons1. Attenuation of glutamate-induced increase in [Ca²⁺ ]_i by 17β-estradiol1.1 Effect of 17β-estradiol on glutamate-induced increase in [Ca²⁺]_iExperiments were performed at room temperature according to the following protocol except those especially indicated: cultured hippocampal neurons were perfused with Mg²⁺-free BSS for 1min, the perfusion rate was 3ml/min. Administration of glutamate (300μmol/L) in the perfusate for lmin resulted in a significant increase in [Ca²⁺ ]_i. After a rinse with Mg²⁺-free BSS for 2.5min, the neurons were then preincubated with 17β-estrsdiol (5×10^-5mol/L) for 3min, the following glutamate administration resulted in a smaller glutamate-induced increase in [Ca²⁺]_i. than that before 17β-estradiol application by 43.2±2.1% in 43 out of 46 neurons (93%). Fitting of the concentration-response curve gives rise to an IC₅₀ of 1.8μmol/L.The inhibitory effect of 17β-estradiol on glutamate-stimulated [Ca²⁺ ]_i increase could be washed out.1.2 Effect of estradiol-BSA on glutamate-induced [Ca²⁺ ]_i increaseEstradiol-bovine serum albumin (E2-BSA), which does not enter the cells, was used to determine whether the effect of estradiol was carried out by a non-genomic mechanism on the cell membrane. E2-BSA at 5×10^-5mol/L reduced the glutamate-induced [Ca²⁺ ]_i increase by 42.9±6.1% in 44 out of 46 neurons (96%). Both 17β-estradiol and E2-BSA showed similar potency in reducingglutamate-stimulated increase in neuronal [Ca²⁺ ]_i. 1.3 Antagonism of 17β-estradiol or estradiol-BSA -induced effect by ICI 182,780To examine the role of estrogen receptor (ER) in the actions of 17β-estradiol, a specific ER antagonist ICI 182,780 was used. Exposure of the neurons to ICI 182,780 (10^-6 mol/L) abolished the inhibitory effect of 17β-estradiol and E2-BSA on glutamate-stimulated increase in neuronal [Ca²⁺ ]_i.2. Effect of 17β-estradiol on β-amyloid_25³5(Aβ)-induced neuronal damageMTT assay was used to measure the effect of 176-estradiol on Aβ_25-35 -induced lowering of the neuronal viability. In the cultured hippocampal neurons, administration of Aβ_25-35 for 24h decreased the neuronal viability. Preincubation of 17β-estradiol (10^-7, 10^-6mol/L) for 72h significantly attenuated the effect of Aβ_25-35. ICI 182,780 (10⁶ mol/L) abolished the protective effect of 17β-estradiol. However, membrane impermeable E2-BSA did not exert the protective effect of 17β-estradiol.Part II Effect of raloxifene on glutamate-induced increase in [Ca²⁺ ]_i and voltage-dependent calcium current1. Effect of raloxifene (low concentration) on glutamate-induced increase in [Ca²⁺ ]_i and voltage-dependent calcium current1.1 Effect of raloxifene on glutamate-induced increase in [Ca²⁺ ]_iIncubation of rat cortical neurons with 10^-5mol/L of raloxifene for 3min, reduced glutamate-stimulated increase in [Ca²⁺]_i by 35.0±3.4%. Raloxifene (10^-7～10^-5mol/L) showed an inhibitory effect on glutamate-induced [Ca²⁺]_i increase in a concentration-dependent manner.The inhibitory effect of raloxifene on glutamate-stimulated [Ca²⁺]_i increase could be washed out.The estrogen receptor antagonist ICI 182,780 (10^-6 mol/L) did not affect the inhibitory action of raloxifene on glutamate-stimulated increase in neuronal [Ca²⁺]_i, implying that the effect was not ER-dependent.As a blocker of internal calcium uptake into endoplasmic reticullum calcium stores, thapsigargin was extensively used as a calcium stores depletionagent. After preincubaion of neurons with 10^-6 mol/L thapsigargin for 25min, the intracellular calcium stores was believed to be depleted completely, however, the action of raloxifene on glutamate-stimulated increase in neuronal [Ca²⁺]_i still existed, indicating that the effect of raloxifene was not dependent on the function of endoplasmic reticullum.1.2. Effect of raloxifene on voltage-dependent calcium currentBy using whole-cell patch clamp, the calcium current was recorded. Incubation of the neurons with 10^-5mol/L of raloxifene for 5min, the high voltage-dependent calcium current was reduced by 50.97±9.51%.2. Effect of raloxifene (high concentration) on glutamate-induced increase in [Ca²⁺ ]_i Incubation of the cortical neurons with raloxifene (2.5×10^-5～7.5×10^-5 mol/L) reduced the basal [Ca²⁺]_i in a concentration-dependent manner. This effect of raloxifene could also be washed out.In summary, the above-mentioned results demonstrate that:1. 17β-estradiol acutely reduces glutamate-stimulated intracellular Ca²⁺ increase via ERs probably on the cell surface of the hippocampal neurons.2. 17β-estradiol reduced Aβ_25-35 -induced neuronal viability decreasion probably mainly via classical cytosolic/nuclear estrogen receptors.3. Raloxifene (10^-7～10^-5 mol/L) reduces glutamate-stimulated intracellular Ca²⁺ increase. This action is acute, non-genomic, and independent of ICI 182,780-sensitive estrogen receptors. High voltage-dependent calcium channel is possibly involved in the underlying mechanisms.4. Raloxifene (2.5 ×10^-5～7.5×10^-5 mol/L) has a reductive effect on [Ca²⁺]_i baseline in a concentration-dependent manner. This action is possibly carried out by a non-genomic mechanism.